Document jm54oKko6Gb0pVgKn243jXbk

CONFIDENTIAL AR226-3127 DPT 432/985069/SS SKIN SENSmZATION TO THE GUINEA-PIG (MAGNUSSON & KLIGMAN METHOD) Sponsor DuPont Specialty Chemicals Jackson Laboratory. Chambers Works Deepwater NJ 08023 USA Research Laboratory Huntingdon Life Sciences Ltd P 0 Box 2 Huntingdon Cambridgeshire PE186ES ENGLAND Report issued 27 July 1999 Page 1 of 25 Compan.y, Sar^ed. Goes not contain TSCACE^ CONTENTS DPT 432/985069/SS Page COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS..................... 3 QUALITY ASSURANCE STATEMENT........................................................................... 4 RESPONSIBLE PERSONNEL......;.................................................................................... 5 6 SUMMARY.............;...................................................................................................-..... 7 INTRODUCTION.............................................................................................................. TEST SUBSTANCE........................................................................................................... 8 EXPERIMENTAL PROCEDURE...................................................................................... 9 15 RESULTS.................................................................................................................... 16 CONCLUSION......................................................-..........-...........................---............ FIGURE 1. Position of intradermal injections and topical induction application............................. 17 TABLES 1. Dermal reactions observed after each induction .......................................................... 18 2. Dermal reactions observed after the challenge application........................................... 19 APPENDICES 1. Individual bodyweights 21 --....--.--..--.,....--....--..--...--......,...-------..-.....--.------.--. 2. Results of preliminary investigations .......................................................................... 22 3. Summary of positive control data............................................................................... 24 Company --San--it'.z;e--^1- D^pce-3s not contain TSC-ACB ^ ^" DPT 432/985069/SS COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS ^?- i?6^ 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. a^' i^: <;^ " -- ^t?-^,." The United Kingdom Good Laboratory Practice Regulations 1^^7-(Statntoiyihstrument--~ No 654). EC Council Directive 87/18/EEC of 18 December 1986 (Official Journal No L 15/29) and. from 1 May 1999. EC Commission Directive 1999/1 I/EC of 8 March 1999 (Official Journal No L 77/8). OECD Principles of Good Laboratory Practice (as revised in 1997) ENV/MC/CHEM(98)17. Illa^^infune with normal practice in this type of short term study, the protocol did not require chemical li"^': analysis of formulated test and control articles for determination of stability, homogeneity and Sl^r ."concentration. . ''^>^.. .-^^^M.-t?^ David G. Coleman, B.Sc. (Hons.), Study Director, Huntingdon Life Sciences Ltd. : -> : Company Sanitized. Doss not contain TSCA CBi QUALITY ASSURANCE STATEMENT DPT 432/985069/SS The following have been inspected or audited in relation to this study Study Phases Inspected Protocol Audit Date of Inspection 20 October 1998 Process Based Inspections Housing/Environment Husbandry Test Material:Control/Administration Treatment Procedure Scoring ..Records Audit Training Records Report Audit 13 August 1998 13 August 1998 13 August 1998 13 August 1998 13 August 1998 13 August 1998 13 August 1998 8 January 1999 Date of Reporting 20 October 1998 14 August 1998 14 August 1998 14 August 1998 14 August 1998 14 August 1998 14 August 1998 14 August 1998 13 January 1999 Protocol Audit: An audit of the protocol for this study was conducted and reported to the Study Director and Company Management as indicated above. Process based inspections: At or about the time this study was in progress inspections and 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. h .1 '^."s*?;1"..... .c?^o^%.................. Margaret Blows, Quality Assurance Group Leader, Department of Quality Assurance, Huntingdon Life Sciences Ltd. 3. <?....7?;-^. W^. Date : 4 Company SanISSe-ssL Doe's no! contain TSCA CB! RESPONSIBLE PERSONNEL David G. Coleman B.Sc. (Hons.), Study Director, Department of Acute Toxicology. DPT 432/985069/SS :5 : Compsny SanitiEod. "323 not contajn T3CA CBi SUMMARY DPT 432/985069/SS This study was performed to assess the skin sensitization potential ofqJ||^^BRusing the guinea- pig. The method followed was that described in: EEC Methods for the determination of toxicity. Annex to Directive 96/54/EC (Official Journal No. L248, 30.9.96), Part B. Method B.6. Skin sensitization; OECD Guideline for Testing of Chemicals No. 406 "Skin Sensitization". 1992. Adopted: 17 July MAGNUSSON, B. And KLIGMAN, A.M. (1970) Allergic Contact Dermatitis in the Guineapig: Identification of contact allergens, Thomas, C.C., Springfield, Illinois, U.S.A. The guinea pigs were dosed by intradennal injection and topical application as these are the routes of exposure required by the test guideline and method. Based on the results of a preliminary study and in compliance with the guideline, the following dose levels were selected: Intradennal injection: 0.25% v/v in water for irrigation Topical application: as supplied Challenge application: 50 and 25% v/v in distilled water Ten test and five control guinea-pigs were used in this study. iHUBBB In this study d1^ not produce evidence of skin sensitization (delayed contact nypersensitivity) in any of the ten test animals. Q^jl^BS--ioes not require labelling with the risk phrase R43 "May cause sensitization by skin contact" in accordance with Commission Directive 93/2 I/EEC. 6 : Company Sanitized. Doss not contasn TSCA C5* ^tg^.-^^-'.'?-- ' INTRODUCTION DPT 432/985069/SS IISglflaausstudy was designed to assess the skin sensitization potential of sing the guinea-pig. ^^^FoUowinginitial exposure to the test substance (the 'induction' period comprising intradennal injections iand'topicaal pplication) the animals were subjected;sppF^amately-twe-wegks-after-fee-topical inductioa^exposure, to a 'challenge' exposure of the test substance in order to establish if a hypersensitive state ^HaS-been induced. Sensitization is determined by examining the skin reaction of test animals to the t|||||E|ehaIlengeexposure in comparison to skin reactions demonstrated by control animals. ' ^tS^^'-ft^^i^.^'. ' ' . ^^I^Hfhe^test substance may come into contact with skin during handling or use. ' ii^ip,..:^ie;-study was conducted in compliance with: ySiSSSK^.'' EEC Methods for the determination of toxicity, Annex to Directive 96/54/EC (Official "^%%Sss-" No. L248, 30.9.96), Part B, Method B.6. Skin sensitization. SisS^--.||@C' ' OECD Guideline for Testing of Chemicals No. 406 "Skin Sensitization". Adopted: ^^^^j^" ^--^---i OQ'? Journal 17 July S-SK-SSi^iS--^'.' - sS^r--Fhs method used was the guinea-pig maximisation ^71;:^:KE^GMAN,A.M. (1970) Allergic Contact Dermatitis 'allergens, Thomas, C.C., Springfield, Illinois, U.S.A. test described by in the Cjuinea-pig: MAGNUSSON, Identification of B. and contact :On this occasion ten test and five control animals were used. iV^The albino guinea-pig was chosen as the test species as it had been shown to be a suitable model for ^gprj^kin' sensitization studies and is the species recommended by the test guidelines. |g@;|^The dose levels for the study were chosen on the basis of a preliminary study in compliance with the Sp^'^'igiiideline. ' The protocol was approved by Huntingdon Life Sciences Management on 7 July 1998, by the Sponsor l^.-.:^-..on 17 ^ly ^98 and by the Study Director on 14 October 1998. :--- -The experimental phase of the study was undertaken between 20 October and 28 November 1998. :7 : .Sanitized. Does not contain TSCA C31 Company Identity: "Chemical name: Intended use: Appearance: Storage conditions: Lot number: Expiry: Purity: Date received: TEST SUBSTANCE DPT 432/985069/SS Two years from date of receipt 23 June 1998 8: Company ,San-it.i-z.e."., ouoee"-rr.oot contain TSCACBi EXPERIMENTAL PROCEDURE DPT 432/985069/SS MANAGEMENT ^^^^^n^ healthy female (which were nulliparous and non-pregnant) albino ~"'"" ' -Jey strain were obtained from D. Hall, Newchurch, Staffs, UK. guinea-pigs of the ^^fe?animalswere approximately four to seven weeks of age on arrival and were acclimatised to the g^enmental environment for six days prior to the start of the main study. The guinea-pigs were within fg^lgltBeaveight range 343 - 429 g at the start of the study (Day 1). ^^fl^Sflditional six animals from the same supplier were used for the preliminary investigations. ^^^^N1;^^' F-s>"^:->'=---!^S5'^.--^.5* *:--. ^|S^rnals on the main study were aUocated without conscious bias to two groups as follows: Group Control animals Test animals Number of animals 10 Animal numbers 4895 to 4899 4900 to 4909 II^^Ihe guinea-pigs were housed in groups of five in suspended metal cages with wire mesh floors in rt^i|Btiilding R17 Room 13. ||J^^:vitaCmenirniched guinea-pig diet (Harlan Teklad 9600 FD2 SQC) and drinking water were ^S^provided ad libifum. Hay was given thrice weekly. ^ ^ | T ^ batch of diet used for the study was analysed for nutrients, possible contaminants or micro- |^6irgamsms, likely to be present in the diet, and which, if in excess, may have had an undesirable effect ^n'^oiOhe test system. The certificates of analyses were lodged in Huntingdon Life Sciences Limited ^' . Archives. There were no known contaminants present in the diet which were expected to be capable of jS^^iPterfering with the study outcome. '-::'-^/. . . ^7 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. Animal,room temperature was controlled within the range 16 - 26.5':'C and relative humidity within the -range 38-68%. These environmental parameters were recorded daily. Lighting was controlled by means of a time switch to give 12 hours of artificial light (0700 - 1900 hours) in each 24 hour period. : 9 Compsny Sanitized. Doss not contain T3CA CB? K^^-J; DPT 432/985069/SS Jtchanimal was identified by ear tattoo number. This number was unique within the Huntingdon Life teicienceAscute Toxicology Department throughout the duration of the study. Each cage was identified %iS^. coloured label displaying the study schedule number, animal numbers and the initials of the Study "ect6r and Home Office licensee, ttSITIVE CONTROL -.lite-seasitivity of the guinea-pig strain used is checked periodically at Huntingdon Life Sciences with fe^nown^sensitisehresxyl cinnamic aldehyde (HCA), Benzocaine and 2-mercaptobenzothia2ole (MET). ^^^Tesults of recent tests are presented in Appendix 3. 5".?5'-?-.''?'- - , sIT^SUBSTANCE PREPARATION lEK^testsubstance was prepared prior to each application on the day of dosing in water for irrigation Sllmtradermainljections) and distilled water (topical application). The concentrations used are described llllin'sthe.treatment procedure. w^-^-- ^%TKe absorption of the test substance was not determined. -,,,M;----7-~-:;-- ---"" ^seg}K^:,>.; .. [^K^Sffie-homogeneity,stability and purity of the test substance were the responsibility of the Sponsor. ^ggTREATMENT PROCEDURE lllig^Preliimnary study Egsiisg^^ ' ^^^jilTfieintradermal and topical imtancy of a range of dilutions of the test substance was investigated to l|Sj|^identuy where possible (a) concentrations of the test substance that would produce irritation suitable for Ipl^ptfie induction phase of the main study and (b) a maximum non-irritant concentration by the topical route j&l^y'bf administration for the challenge phase. The animals for the topical imtancy investigations were pre-treated with an mtradermal injection of g^^-freund's complete adjuvant, 50 : 50 with water for irrigation' (Ph.Eur.), approximately one week prior ^k^L.L.totne start of the preliminary investigations. ^.^ The numerical values given to the dermal reactions observed in the preliminary tests are shown in a^.;-;,. Appendix 2. CY- ; IJ^J Sterile water for injection ^n : 10 : . . ^ Conpany Sanded. SCACBS not conta-^ DPT 432/985069/SS Selection of concentrations of test substance for the main study Based on the results of the preliminary investigations, the following concentrations of j^were selected: Induction intradennal injection - 0.25% v/v in water for irrigation This was the highest concentration that-caused initatkm but did-no^ adversely affect-the aoifflals. Induction topical application - as supplied 1111^-. The test material applied topically as supplied produced some irritation but did not adversely affect the animals.' Jiff^'. Topical challenge - 50 and 25% v/v in distilled water S'"'!!;;^^^. aSSi:- From preliminary investigations 50% v/v in distilled water was the highest concentration not |i|sE '. giving rise to irritating effects. fT?.'-^- ::-'. H^'"- Main study E^B---- e^'"' ' ^L::. The procedure may be considered in two parts. Induction and Challenge. |&: te: - Induction Induction intradennal injections - test animals A 40 x 60 mm area of dorsal skin on the scapular region of the guinea-pig was clipped free of hair with electric clippers. Three pairs of intradennal injections were made into a 20 x 40 mm area within the clipped area as shown in Figure 1. Injectables for the test animals were prepared as follows: 1. Freund's complete adjuvant** was diluted with an equal volume of water for irrigation (Ph.Eur.). 2. |1^BHUo.25% v/v in water for irrigation. 3. ^IHBHiBB-^0^ v^ in a 50 : 50 mixture of Freund's complete adjuvant and water ** Difco Laboratories, Detroit, Michigan, U.S.A. 11 : ^-".-'.-3^cor;ta:nTSC&CB8 <~.w^>->nv oSs's-'.ii!'--iai.reiu-'-----' " DPT 432/985069/SS Induction topical application - test animals One week after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free of hair. ^ B B U H F A 20 x 40 mm patch of Whatman No. 3 paper was saturated with approximately 0.4 ml of supplied. The patch was placed on the skin of the test animals and covered by \ length of impermeable plastic adhesivertaper (50^nnn width "Blenderm"). This- in turn- was firmly secured by elastic adhesive bandage (50 mm width "Elastoplast") wound round the torso of the animal and fixed with "Sleek" impervious plastic adhesive tape. The dressing was left in place for 48 hours. Induction - control animals During the induction phase, the control animals were treated similarly to the test animals with the exception that the test substance was omitted from the intradermal injections and topical application. The dermal reactions observed after each induction phase in both control and test animals are shown in Table 1. Challenge Challenge - control and test animals The control andtestanimalswere challenged topically two weeks after the topical induction application usingHUUHQ50 and 25% v/v in distilled water. Hair was removed by clipping and then shaving from an area on the left flank of each guinea-pig. __ A 20 x 20 mm patch of Whatman No. 3 paper was saturated with approximately 0.2 ml of H^HlB|50% v/v in distilled water and applied to an anterior site on the flank. ^|B25% f J H v/v in distilled water was applied in a similar manner to the posterior site. The patches were sealed to the flank for 24 hours under strips of "Blendenn" covered by "Elastoplast" wound round the trunk and secured with "Sleek". OBSERVATIONS Clinical signs All animals were observed daily for signs of ill health or toxicity. I^S^-'^Bodyweight 1?% |s -:,: '^le bodyweight of each guinea-pig on the main study was recorded on Day 1 (day of intradermal "yections) and on the last day observations were made of dermal responses to the challenge application. 12 : Company Sar-^zsd. Doss not contain TSCA CS- DPT 432/985069/SS Dermal responses The dermal reactions resulting from intradennal injection and topical application on the preliminary study, and topical application at the challenge were assessed using the following numerical system: Erythema and eschar formation: No erythema 0 Slight erythema 1 Well-defined erythema 2 Moderate erythema 3 Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4 Oedema formation: No oedema 0 Slight oedema 1 Well-defined oedema (edges of area well-defined by definite raising) 2 Moderate oedema (raised approximately 1 millimetre) 3 Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) 4 The approximate diameter (mm) of the dermal response at the intradennal injection sites was recorded in the preliminary study only to assist in the choice of concentrations for the main study. * Any lesion not covered by this scoring system was described. The challenge sites were evaluated 24 and 48 hours after removal of the patches. On completion of the study all animals were killed by cervical dislocation. INTERPRETATION OF THE RESULTS Dermal reactions in the test animals elicited by the challenge application were compared with the findings simultaneously obtained in the control animals. A test animal was considered to show positive evidence of delayed contact hypersensitivity if the observed dermal reaction at challenge was definitely more marked and/or persistent than the maximum reaction seen in animals of the control group. It the dermal reaction seen in a test animal at challenge was slightly more marked and/or persistent than (but not clearly distinguishable from) the maximum reaction seen in control animals, the result for that test animal was classified as inconclusive. A test animal was considered to show no evidence of delayed contact hypersensitivity if the dermal reaction resulting from the challenge application was the same as, or less marked and/or persistent than the maximum reaction seen in animals of the control group. : 13 : Compsrsy Sar.'t^ed. Doss not contain T?CA C3? DPT 432/985069/SS &*. ARCHIVES All raw data and study related documents generated during the course of the study at Huntingdon Life Sciences, together with a copy of the final report will be lodged in the Huntingdon Life Sciences Ltd Archive, Huntingdon. Such records will be retained for a minimum period of five years from the date of issue of the final report. At the end of the five year retention period the Client will be contacted and advice sought on the future requirements. Under no circumstances will any item be discarded without the Client's knowledge. ^ E^;-:' " II:' DEVIATIONS FROM PROTOCOL _ There were no deviations from the protocol that were considered to have affected the integrity or validity of the study, however the following is of note: On occasion the temperature of the animal room was outside the range given in the protocol, however this was not considered to have had an adverse effect on the animals. 14 Company Samfesd. Does no? contain TSCA C31 RESULTS DPT 432/985069/SS CLINICAL SIGNS No signs of ill health or toxicity were observed. BODYWEIGHT Individual bodyweights are shown in Appendix 1. Bodyweight increases were recorded for all guinea-pigs over the period of the study: INDUCTION Dermal reactions seen following the induction applications are summarised in Table 1. Intradermal injections Necrosis was recorded at sites receiving Freunds Complete Adjuvant in test and control animals. Slight irritation was seen in four test animals at sites receivingHUUR 0.25% v/v in water for irrigation and slight irritation was observed in one control animal receiving water for irrigation. No reactions were noted for the remaining test or control animals. Topical application Slight erythema was observed in most test animals following topical application wit supplied. Slight erythema was seen in one control guinea-pig. CHALLENGE ' The numerical values given to the dermal reactions elicited by the challenge applications are shown in Table 2, There were no dermal reaction? seen in any of the test or control animals, therefore all ten test animals gave negative responses. : 15 : Company Sanded. Does not c^tai" TSCA CBs CONCLUSION DPT 432/985069/SS In this study, IH^^BH^ did not produce evidence of skin sensitization (delayed contact hypersensitivity) in any of the ten test animals. 16 : Company San^ri. Does not contain TSCA CE? DPT 432/985069/SS FIGUREl Position of intradermal injections and topical induction application The rectangle outlines the 20 x 40 mm clipped scapular area in which injections were made and to which the topical induction application was made one week later. Control animals: (1) 0.1 ml of Freund's complete adjuvant 50:50 with water for irrigation (Ph.Eur.). (2) 0.1 ml of water for irrigation. (3) 0.1 ml ofFreund's complete adjuvant 50 : 50 with water for irrigation. Test animals: (1) 0.1 ml of Freund's complete adjuvant 50:50 with water for irrigation (Ph.Eur.). (2) 0.1 ml orBHHIIHI 0.25% v/v in water for irrigation. (3) 0.1 ml o complete adjuvant. [ 0.25% v/v in a 50 : 50 mixture of water for irrigation and Freund's A volume ofO.l ml was injected into both the left and right injection sites. 17 : Company Saniteed. Do- -- ^te^ T3CA ^ TABLE 1 DPT 432/985069/SS Dermal reactions observed after each induction Group Control Test Animal number 4895 4896 4897 4898 4899 4900 4901- - 4902 4903 4904 4905 49064907 4908 4909 Intradennal injections Site number 1.2 3 '-"."N" 0 N N 0 N N 1 N N 0 N . NO N N 0 N ----N------0 N N 0 N N 1 N N 1 N N 0 N N - 1 N N 0 N N 1 N N 0 N Topical application 0 0 1 0 0 0 1 1 1 1 1 0 1 1 1 Intradennal injections Topical application Control animals: See figure 1 (previous page) Test animals: See figure 1 (previous page) Control animals: disUlled water Test anunals:UUBHH^as supplied N Necrosis 0 No irritation 1 Slight irritation 2 Well-defined irritation 3 Moderate irritation 4 Severe irritation 0 No erythema 1 Slight erythema 2 Well-defined erythema 3 Moderate erythema 4 Severe erythema 18 Company Sanitsed. DG'-S nat c3n<ain TSCA CES TABLE 2 DPT 432/985069/SS Dermal reactions observed after the challenge application -with Freund's treated controls Guinea-pig number E = Erythema 0 = Oedema 4895 E 0 4896 E 0 4897 E 0 4898 E 0 4899 E 0 Anterior site, exposed ti Posterior site. exposed Score - --- -..--:-- .-.--.-. - -v:7--.r.^ 24 Hours A P 0 0 0 0 48 Hours :'-- ? --- - A- - -.-p----< -----. 0 . 0 :.--: 0 "'" '.'(L.'.C s:^:^---.. - 0 0 0 0 0 : 0 0 0 0 . 0 0 0 "6""" -------------- ooo0 0 ,- -.. -.-.-..-. 00000000 000 000 50% v/v in distilled water (25%v/v in distilled water : 19 Comp-a..n.ySr-it.z.''."""'0'""1""7^081 TABLE 2 Dermal reactions observed after the challenge application with| (continued) DPT 432/985069/SS Test animals Guinea-pig number 4900 4901 4902 4903 4904 4905 4906 4907 4908 4909 E = Erythema 0 = Oedema E 0 E 0 E 0 E 0 E 0 E 0 E 0 E 0 E 0 E 0 Anterior site, exposed Posterior site, ex-posed ti ---------Seore -- 24 Hours 48 Hours A P A P 0 0 0 0 0 0 .-0. - 0 0 0 00 0 0 0000 0000 0 0 0000000000000000000000000000 ,50%v/vm distilledwater 25% v/v in distilled water - Results Positive (+) Negative (-) Inconclusive () - - - - - - - - - - ^co^s^."-""^06' Group Control Test. APPENDIX 1 Individual bodyweights (g) DPT 432/985069/SS Guinea-pig number 4895 4896 4897 4898 4899 4900 4901 4902-- : 4903 4904 4905 4906 4907 4908 4909 Day 1 4 November 1998 . 343 406 387 362 379 370 359 375 365 368 361 348 404 379 429 Last observation day 28 November 1998 514 627 544 540 579 588 537 555 555 547 527 546 628 533 672 : 21 :Compsny Sanitized. Does not contain i SCA CBI APPENDIX 2 DPT 432/985069/SS Results of preliminary investigations withi Intraderinal injections Vehicle: Water for irrigation Guineapig number Concentration %v/v Score 4449 10.0 Hours 24 72 . D 10 8 E N N 0 2 . .2 7.5 D 10 10 E N N 0 2 2 5.0 D 10 8 E N N 0 2 2 2.5 D 8 8 E N N 0 2 2 1.0 D 8 8 E SN N 0 2 2 0.5 D 8 8 E SN SN 0 2 2 0.25 D 8 8 E 2 2 0 2 2 0.1 D 6 8 E 2 2 0 2 2 Vehicle control D 6 8 E 2 2 0 2 2 --GuiaeaPig number 4450 Concentration ---.---Score---. .... %v/v 10.0 7.5 5.0 2.5 1.0 0.5 0.25 0.1 Vehicle control Hours D E 0 D E 0 D E 0 D E 0 D E 0 D E 0 D E 0 D E 0 D E 0 24 72 10 10 N N 2 2 10 10 N N 2 2 8 8 N N 2 2 8 8 ' SN N 2 2 8 8 SN N 2 2 8 8 SN SN 2 2 8 8 2 2 2 2 6 8 2 2 2 2 6 6 2 2 2 2 Key: D Diameter (mm) E .Erythema (0-4 numerical scores) 0 Oedema (0-4 numerical scores) SN Slight necrosis N Necrosis : 22 : Company S^feccS. Do-s not contain TSCA CBS APPENDIX 2 Results of preliminary investigations with (continued) TopicaT-application DPT 43 2/985 069/SS Vehicle: distilled water Score Guinea-pig Concentration number % v/v 0 Hours;-:.:'"- '24Hours g ... ^..Q.--.,-..-.-g---..-..Q- 48 Hours g Q TA-AtUfiUfi 75 ---11------ -----1 i---- --.*--A.i.,.--- .*-(:U -. - -Li j ]s * o U - " " ' 50 0" 0 "O"""' 0 0 0 . 4467 4470 4471 4761 4762 25 10 75 50 25""" 10 75 50 25 10 75 50 25 10 100 90 100 - % 100 0-0 000 0 0 0 0 0 0 0 1 0 1 0 I* 0 ' 0 0 0 0 0 0 000000 1 0 1 0 I* 0 000000 0 0 0 0 0 0000000000 1 0 1 0 O* 0 0 0000 0 0 0 0 000001000000000001000000000000000000 1 0 L1 0 0 0 90 1 100 0 90 0 E Erythema (0-4 umueriual scores) 00 0 0I* I* 00 0 00 0 0 0 0 0 Oedema (0-4 numerical scores) L Localised dermal reaction * Dryness and sloughing ofd the epidermis 23 Company Sanitized. Does not coiUain TSCA C3i DPT 432/985069/SS APPENDIX 3 Skin sensitization positive control study with 2-Mercaptobenzothiazole (MBT) to the Magnusson & Kligman method (Sch. No. HLS/010) This study was perfbnned to confirm the sensitivity and reliability of the experimental technique used at Huntingdon Life Sciences to detect skin sensitization potential. ..The study was performed using the guinea-pig and a known weak/moderate sensitizer-- Z-Mcrcaptobchzothiazolc (MBT^:--The-method followed was that described in: MAGNUSSON, B. and KLIGMAN, A.M. (1970) Allergic Contact Dermatitis in the Guineapig: Identification of contact allergens, Thomas, C.C., Springfield, Illinois, U.S.A. This positive control study was conducted-befween-23"June"and 17 July 1998 using fifteen female (nulliparous and non pregnant) albino guinea-pigs of the Dunldn Hartley strain supplied by D Hall, Staffs, UK. The MBT used for this study Was a pale yellow powder, batch number 16H3435, expiry date 3 April 2000, supplied by Sigma Chemicals St Louis, USA. Based on preliminary investigations previously conducted at this laboratory, the following concentrations of MBT were administered: Intradermal injection: Topical application: Challenge application: 10 % w/v in AJembicol D 83.3% w/v in Alembicol D 83.3 and 40% w/v in Alembicol D RESULTS INDUCTION Intradermal injections - Slight to MBT, 10% w/v in Alembicol D AJembicol D. well-defined irritation was seen in test animals at sites and slight irritation was observed in control animals receiving receiving Topical application - Well-defined erythema was observed in test animals following topical application with MBT, 83.3% w/v in Alembicol D. Well-defined erythema was seen in the control animals receiving Alembicol D. CHALLENGE Dermal reactions were noted in all of the ten test animals compared to none in the controls therefore all ten test animals were considered to have given positive sensitization responses. CONCLUSION In this study MET produced evidence of skin sensitisation (delayed contact hypersensitivity)in all of the ten animals, thus confirming the sensitivity and reliability of the experimental technique. Company Sanded. 5oes not contain TSCA C31 DPT 432/985069/SS APPENDIX 3 (continued) Individual dermal reactions after challenge application of MBT CONTROL ANIMALS Guinea-pig E = Erythema Score number 0 - Oedema 24 Hours 4s Hours A P A P 2460 2461 0000000 0 2462 2463 000000000000 2464 E 0 0 0 E 0 0 0 0 - -.--0------..-,0-.- 0 0E .0-..- ,--0,..,-- -0 0 E 0 E 0 0 0 0 0 TEST ANIMALS Score Results Guinea-pig E = Erythema Positive (+) number 0 = Oedema 24 Hours 48 Hours Negative (-) A P A P Inconclusive () 2465 E 2 1 02 01 + 0 2466 E + 0 1 0 I* 0* 1 10 1 10 2467 E + 0 0 1 0 I* 0 1 0 1 2468 E 1 1 01 01 + 2469 1 0 1 1 2470 1 0 1 0 2471 2472 2 2 2 1 2473 .V 1 0 1 0 2474 0 E 0 E 0 E 0 E 0 E 0 E 0 1 1 01 01 + 2 2 02 NE2 + 2 2 2 02 + 2 1 I* 0 2 1 02 01 + 2 2 02 2 + + 2 2 2* 2* 212212 * Dryness and sloughing of the epidermis 0 Thickening, dryness and sloughing of the epidermis k) NE Necrotic edge A Anterior site, exposed to MET, 83.3% w/v in Alembicol D P Posterior site, exposed to MBT, 40% w/v in Alembicol D : 25 Compare Sanitized. Goes not contain TSCA C3E