Document RjmdYg8yeK4VKqwZ5vee1pNwv

CONFIDENTIAL AR226-3127 DPT 432/985069/SS SKIN SENSITIZATION TO THE GUINEA-PIG (MAGNUSSON & KLIGMAN METHOD) Sponsor DuPont Specialty Chemicals Jackson Laboratory. Chambers Works Deepwater NJ 08623 USA Research Laboratory Huntingdon Life Sciences Ltd P O Box 2 Huntingdon Cambridgeshire PE 18 6ES ENGLAND Report issued 27 July 1999 Page 1 of 25 Compsny anitized . Does not contain TSCA CE? CONTENTS DPT 432/985069/SS Page COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS....................... 3 QUALITY ASSURANCE STATEM ENT.............................................................................................. ^ RESPONSIBLE PERSONNEL....................................................................... 5 SUMMARY............. :......................................................................................... IN T R O D U C T IO N ............................................................................................. TEST SUBSTANCE.......................................................................................... EXPERIMENTAL PRO CED U RE................................................................. 6 7 8 9 RESULTS........................................................................................................ .. CONCLUSION......................................................................... ........................ 15 16 FIGURE 1. Position of intradermal injections and topical induction application 17 TABLES 1. Dermal reactions observed after each induction.......... .................... 2. Dermal reactions observed after the challenge application............... 18 19 APPENDICES 1. Individual bodyweights............................ 2. Results of preliminary investigations .... 3. Summary of positive control d ata......... :2 : Company 0 3nitizc ces not c( ntain TSCACB DPT 432/985069/SS COMPLIANCE WITH GOOD LABORATORY PRACTICE STANDARDS The study described in this report was conducted in compliance with the following Good Laboratory Practice Standards and with the exception o f that noted below I consider the data generated to be valid. ~ The United Kingdom Good Laboratory Practice Regulations 4997 No 654). - ... EC Council Directive 87/18/EEC o f 18 December 1986 (Official Journal No L 15/29) and, from 1 M ay 1999, EC Commission Directive 1999/11/EC o f 8 March 1999 (Official . Journal No L 77/8). OECD Principles o f Good Laboratory Practice (as revised in 1997) ENV/MC/CHEM(98)17. In line with normal practice in this type of short term study, the protocol did not require chemical analysis of formulated test and control articles for determination o f stability, homogeneity and concentration. David G. Coleman, B.Sc. (Hons.), Study Director, Huntingdon Life Sciences Ltd. Company Sanitized. Doss not contain TSCA CBS 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 o f study were earned out. These were conducted and reported to appropriate Company Management as indicated above R eport 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 Margaret Blows, * Quality Assurance Group Leader, Department o f Quality Assurance, Huntingdon Life Sciences Ltd. Date :4 : Company Sanitized.' Does no! contain TSCA C3 RESPONSIBLE PERSONNEL David G. Coleman B.Sc. (Hons ), Study Director, Department o f Acute Toxicology. DPT 432/985069/SS :0 : Company Sanitized. Doss no? contain T3CA CBi SUMMARY DPT 432/985069/SS This study was performed to assess the skin sensitization potential o pig. The method followed was that described in: [using the guinea- EEC Methods for the determination o f 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 o f Chemicals No. 406 "Skin Sensitization". Adopted: 17 July 1992. MAGNUSSON, B. And KLIGMAN, A.M. (1970) Allergic Contact Dermatitis in the Guineapig: Identification o f contact allergens, Thomas, C.C., Springfield, Illinois, U.S.A. The guinea pigs were dosed by intradermal injection and topical application as these are the routes o f exposure required by the test guideline and method. Based on the results o f a preliminary study and in compliance with the guideline, the following dose levels were selected: Intradermal 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. In this study did not produce evidence o f skin sensitization (delayed contact hypersensitivity) in any o f the ten test animals. S S ^ S & i o e s not require labelling with the risk phrase R43 "May cause sensitization by skin contact" in accordance with Commission Directive 93/21/EEC. :6 : INTRODUCTION DPT 432/985069/SS ||g j |p jg p '' S ^ P h i s study was designed to assess the skin sensitization potential o sing the guinea-pig. jnitial exposure to the test substance (the `induction' period comprising intradermal injections 7 7 7 7 exposure, to a `challenge' exposure o f the test substance in order to establish if a hypersensitive state t ^ f c r E j ^ l b e e n induced. Sensitization is determined by examining the skin reaction o f test animals to die p p S p j^ en g e exposure in comparison to skin reactions demonstrated by control animals. 7 "'"The test substance may come into contact with skin during handling or use. I ^ ^ P ^ s t a d y was conducted in compliance with: 1*7 EEC Methods for the determination o f toxicity, Annex to Directive 96/54/EC (Official Journal i r : No. L248, 30.9.96), Part B, Method B.6. Skin sensitization. I l l i f OECD Guideline for Testing o f Chemicals No. 406 "Skin Sensitization". Adopted: 17 July .... -1992. : -- The method used was the guinea-pig maximisation test described by MAGNUSSON, B. and ^ H m 7 r ^ K i e M A N , A.M. (1970) Allergic Coniaci Dermatitis in the Guinea-pig: Identification o f contact ' ' - allergens, Thomas, C.C., Springfield, Illinois, U.S.A. ' On this occasion ten test and five control animals were used. . : ' The albino guinea-pig was chosen as the test species as it had been shown to be a suitable model for : skin sensitization studies and is the species recommended by the test guidelines. - ` " The dose levels for the study were chosen on the basis o f a preliminary study in compliance with the " guideline. - The protocol was approved by Huntingdon Life Sciences Management on 7 July 1998, by the Sponsor 5 . on 17 July 1998 and by the Study Director on 14 October 1998. -The experimental phase o f the study was undertaken between 20 October and 28 November 1998. # 7: Company Sanitized. Does not contain TSCA CBl Identity: ' ^Chemical name: Intended use: Appearance: Storage conditions Lot number: Expiry: Purity: Date received: TEST SUBSTANCE DPT 432/985069/SS Two years from date o f receipt 23 June 1998 ! 8 ariitzef! Does not contain TSC CSS Company Sanitize-, ^ mm asp i igipfe! EXPERIMENTAL PROCEDURE DPT 432/985069/SS -ANIMAL MANAGEMENT ^ ^ F i f t e e n healthy female (which were nulliparous and non-pregnant) albino guinea-pigs o f the i3"Si"";r' ~~ J - - -strain were obtained from D. Hall, Newchurch, Staffs, UK. _______________ lie^anim als were approximately four to seven weeks o f age on arrival and were acclimatised to the I f ^ S k p e n m e n t a l environment for six days prior to th e s ta rto f the main study. The guinea-pigs were within j to ffilly b i|h t range 343 - 429 g at the start of the study (Day 1). - - - - a'oT.Lfr-.--''rE'- six animals from the same supplier were used for the preliminary investigations. ^ ^ ^ ^ ^ ^ m a l s on the main study were allocated without conscious bias to two groups as follows: p s iP fc : i: Group Control animals Test animals Number of animals 10 Animal numbers 4895 to 4899 4900 to 4909 The guinea-pigs were housed in groups o f five in suspended metal cages with wire mesh floors in ^--Building R17 Room 13. "A w ta m rn C enriched guinea-pig diet (Harlan Teklad 9600 FD2 SQC) and drinking water were ^provided ad libitum. Hay was given thrice weekly. f f f v fllie' batch of diet used for the study was analysed for nutrients, possible contaminants or micro;y; organisms, likely to be present in the diet, and which, if in excess, may have had an undesirable effect - .......... onTETtest system. The certificates o f 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 . i T i ^interfering with the study outcome. r J-- Results of routine physical and chemical examination o f drinking water, as conducted by the supplier r ~ are made available to Huntingdon Life Sciences Ltd as quarterly summaries. Animal room temperature was controlled within the range 16 - 26.5C and relative humidity within the - range 38-68%. These environmental parameters were recorded daily. Lighting was controlled by - - means o f a time switch to give 12 hours of artificial light (0700 - 1900 hours) in each 24 hour period. :9 : Company Sanitised. Does not contain T3CA CBi DPT 432/985069/SS ^ ^ ^ f e l i b i i c e s Acute Toxicology Department throughout the duration o f the study. Each cage was identified mviiy vi mv no r ^ ^ ^ y ^ S wTi-sensitisers hexyl cinnamic aldehyde (HCA), Benzocaine and 2-mercaptobenzothiazoIe (MBT). ^ ^ ^ S ^ J ^ u i t s o f recent tests are presented in Appendix 3. Ig J ^ n E S T SUBSTANCE PREPARATION test substance was prepared prior to each application on the day o f dosing in water tor irrigation ^ g ^ - ^ t r a d e r m a l injections) and distilled water (topical application). The concentrations used are described i rm the treatment procedure. The absorption o f the test substance was not determined. fe i^ T h e-h o m o g en e ity , stability and purity of the test substance were the responsibility of the Sponsor. TREATMENT PROCEDURE L' - Preliminary study fc fe S a a i- ' - . The mtradermal and topical irritancy of a range o f dilutions of the test substance was investigated to t ~ identify where possible (a) concentrations o f the test substance that would produce irritation suitable for C ' the induction phase of the main study and (b) a maximum non-irritant concentration by the topical route _ " - of administration for the challenge phase. : The animals for the topical imtancy investigations were pre-treated with an mtradermal injection of :' - - j - Freund's complete adjuvant, 50 : 50 with water for irrigation1 (Ph.Eur.), approximately one week prior . to the start o f the preliminary investigations. ." The numerical values given to the dermal reactions observed in the preliminary tests are shown in _ Appendix 2. J 1Sterile water for injection : 10 : Company not contain T5CA v-B. DPT 432/985069/SS Selection of concentrations of test substance for the main study Based on the results o f the preliminary investigations, the following concentrations o: were selected: gltor-f,S,-es3f.scjEjEV';r Induction intradermal injection - 0.25% v/v in water for irrigation This was the highest concentration th at caused iiritation but did n o t adversely affeetthe animals. Induction topical application - as supplied The test material applied topically as supplied produced some irritation but did not adversely affect the animals.' .V Topical challenge - 50 and 25% v/v in distilled water |g |f : From preliminary investigations 50% v/v in distilled water was the highest concentration not giving rise to irritating effects. i l f e . Main study The procedure may be considered in two parts, Induction and Challenge. r y . - Induction Induction intraderm al injections - test animals ' A 40 x 60 mm area o f dorsal skin on the scapular region of the guinea-pig was clipped free o f hair with electric clippers. Three pairs o f intradermal 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. 0.25% v/v in water for irrigation. 0.25% v/v in a 50 : 50 mixture o f Freund's complete adjuvant and water or irrigation. ** Difco Laboratories, Detroit, Michigan, U.S.A. 11 : ( M M * D W > O ' * * 1" TSCA CB< ' ' ' DPT 432/985069/SS vXr " Induction topical application - test animals One week after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free o f hair. A 20 x 40 mm patch of Whatman No. 3 paper was saturated with approximately 0.4 ml o f supplied. The patch wasplaced on the skin o f the test animals and covered by r - - jH e n g t^ ^ n ^ e rm e a b le plastic adhesivertaper (5fr m m width "Blenderm"). This- in tunr 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 and ere challenged topically two weeks after the topical induction application u s i n g H j i H I M Q*50 and 25% v/v in distilled water. Hair was removed by clipping and then shaving from an area on the left flank o f each guinea-pig. A 20 x 20 mm patch of Whatman No. 3 paper was saturated with approximately O ^ n ^ f v/v in distilled water and applied to an anterior site on the flank. ^ ^ 2 5 ^ W / 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 o f Blenderm covered by "Elastoplast" wound round the trunk and secured with "Sleek". OBSERVATIONS > Clinical signs . _ All animals were observed daily for signs o f ill health or toxicity. . : ' ' Bodyweight -- The bodyweight of each guinea-pig on the main study was recorded on Day 1 (day of intradermal \ . injections) and on the last day observations were made o f dermal responses to the challenge application. uissdi* . 12 : Company Sanrtfzsd. Doss not contain TSCA CSi DPT 432/985069/SS Dermal responses The dermal reactions resulting from intradermal 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 Slight erythema Well-defined erythema Moderate erythema Severe erythema (beet redness) to slight eschar formation (injuries in depth) 0 2 3 4 Oedema formation: No oedema Slight oedema Well-defined 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 the area o f exposure) 0 1 2 33 4 The approximate diameter (mm) o f the dermal response at the intradermal injection sites was recorded in the preliminary study only to assist in the choice o f 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 o f the patches. On completion o f 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 o f delayed contact hypersensitivity if the observed dermal reaction at challenge was definitely more marked and/or persistent than the maximum reaction seen in animals o f the control group. . If 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 o f the control group. : 13 : DPT 432/985069/SS a r c h iv e s All raw data and study related documents generated during the course of the study at Huntingdon Life Sciences, together with a copy o f the final report will be lodged in the Huntingdon Life Sciences Ltd Archive, Huntingdon. . _________ !______ Such records will be retained for a minimum period o f five years from the date o f issue o f 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. . 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 o f 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 Sam ites* Does not contain TSCA CBi RESULTS DPT 432/985069/SS CLINICAL SIGNS No signs of ill health or toxicity were observed. b o d y w eig h t Individual bodyweights are shown in Appendix 1. ........... ............ Bodyweight increases were recorded for all guinea-pigs over the period o f the study: I J : : INDUCTION Dermal reactions seen following the induction applications are summarised in Table 1 Intradermal injections Necrosis was recorded at sites receiving Freund's Complete Adjuvant in test and control animals. reactions were noted for the remaining test or control animals. Topical application I--'- Slight erythema was observed in most test animals following topical application wil supplied. Slight erythema was seen in one control guinea-pig. as CHALLENGE The numerical values given to the dermal reactions elicited by the challenge applications are shown in Table 2. There were no dermal reactions seen in any of the test or control animals, therefore all ten test animals gave negative responses. 15 : Com,, ppsa r , S a n i s i - * " TSC& CB' iSwHKS * CONCLUSION DPT 432/985069/SS did not produc evidence o f skin sensitization (delayed contact test animals. 16 Company Sanitized. Doss not coni TSCA CE DPT 432/985069/SS FIGURE 1 Position of intradermal injections and topical induction application ii 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 5 0 : 5 0 with water for irrigation (Ph.Eur.). (2) 0.1 ml o f water for irrigation. (3) 0.1 ml o f Freund's complete adjuvant 5 0 : 5 0 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 (3) 0.1 ml complete adjuvant. 0 -25/0 v/v in water for irrigation. 0.25% v/v in a 50 : 50 mixture of water for irrigation and Freund's A volume o f 0.1 ml was injected into both the left and right injection sites. : 17 : Company Sanitized. Docs ,-io contain T3CA CSt DPT 432/985069/SS TABLE 1 Dermal reactions observed after each induction Group Control Test Animal number 4895 : 4896 4897 4898 .. 4899 4900 4901----- 4902 4903 4904 4905 4906- - 4907 4908 4909 Intradermal injections Site number Topical application 1 .2 ----------- 0______ _N- ..... ................0 ____ N0N 0 N 1N 1 N0N 0 N0N 0 N0 N- 0 N0 N1 N1 N0 N1 N0 N1 N0 N N N N N N N N N N 0 1 1 I 1 1 0 1 1 1 .. Intraderma) injections Topical application Control animals: See figure 1 (previous page) Test animals: See figure 1 (previous page) Control animals: distilled water Test a n i m a l s : ^ m |j ^ |^ ^ a s 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 Sanitized. Do iOt contain TSCA CEi DPT 432/985069/SS TABLE 2 Dermal reactions observed after the challenge application vtitl Freund's treated controls Guinea-pig E = Erythema number O = Oedema 4895 4896 4897 4898 4899 E 0 E 0 E 0 E 0 E 0 A Anterior site, exposed to] P Posterior site, exposed ti Score 24 Hours AP 00 00 00 00 00 00 00 00 00 00 48 Hours''.... A- - -* P -H 00 0 0 ..... ' 00 0 0 :. . 00 00 00 00 00 00 50% v/v in distilled water 25% v/v in distilled water . : 19 : Company Oof is net contain TSCA CBS DPT 432/985069/SS TABLE 2 Dermal reactions observed after the challenge application with (continued) Test animals Guinea-pig number 4900 4901 4902 4903 4904 4905 4906 4907 4908 4909 E = Erythema O = Oedema E O E O E O E O E O E O E O E O E O E O ............... ........ 24 Hours Ap 00 00 00 00 00 00 00 00 0 0 00 00 00 00 00 00 00 00 00 00 00 -- . . . .. - ,, R e s u lts .. Positive (+) 48 Hours Negative (-) A p Inconclusive (+) 00 " 00 00 " 00 00 00 00 - 00 00 " .0 0 0 0 00 00 " 00 00 - 00 00 - 00 00 - 00 A Anterior site, exposed v/v in distilled water P Posterior site, exposed t < n H | ^ ^ H 2 5 % v/v in distilled water : 20 : nlS C A C B i Does not coiA Cmp ry Saairiz e d - Group Control Test. APPENDIX 1 Individuai bodyweights (g) DPT 432/985069/SS Guinea-pig number 4895 : 4896 4897 4898 4899 4900 4901 49024903 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 : Company Sanitize Does not c a n TSCACBi DPT 432/985069/SS APPENDIX 2 Results of preliminary investigations with Intraderirial injections Vehicle: Water for irrigation Guinea- Concentration pig number % v/v 4449 10.0 7.5 5.0 2.5 1.0 0.5 0.25 0.1 Vehicle control Score Hours D E O D E O D E 0 D E O D E O D E O D E O D E O D E O 24 72 10 8 NN 2 . .2 10 10 NN 22 10 8 NN 22 88 NN 22 88 SN N 22 88 SN SN 22 88 22 22 68 22 22 68 22 22 --Guineapig 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 O D E O D E O D E O D E O D E O D E O D E O D E O 24 72 10 10 NN 22 10 10 NN 22 88 NN 22 88 SN N 22 88 SN N 22 88 SN SN 22 88 22 22 68 22 22 66 22 22 Key: -- D Diameter (mm) E Erythema ( 0 - 4 numerical scores) O /' Oedema ( 0 - 4 numerical scores) SN Slight necrosis N Necrosis : 22 APPENDIX 2 Results of preliminary investigations witl (continued) T opicaLapplication DPT 432/985069/SS Vehicle: distilled water Guinea-pig Concentration number % v/v 4466 75 50 25 . 10 4467 75 50 25 ' 10 4470 75 50 25 10 4471 75 50 25 10 4761 100 90 100 - ~ .... 90 4762 100 90 100 90 -_ - : Score 0 Hours. 24 Hours ' E "O " ~E---- "'O ' t .....1 ' '. 1... 1 0 0 0 0. 0 0 000 0 000 l 0 10 0 00 0 0 00 0 0 00 0 i 0 10 0 00 0 0 00 0 0 00 0 1 0 10 0 00 0 0 00 0 0 00 0 1 0 10 0 00 0 0 00 0 0 00 0 1 0 LI 0 100 0 0 0 1* 0 0 0 1* 0 E Erythema ( 0 - 4 numerical scores) O Oedema ( 0 - 4 numerical scores) L Localised dermal reaction * Dryness and sloughing ofd the epidermis 48 Hours E0 LI* 0 00 00 00 1* 0 00 00 00 1* 0 00 00 00 0* 0 00 00 00 00 00 00 00 00 00 00 00 i k.... : rr,, i: fe----' si <.3 I i 1t j` Company Sanitized. Does not contain i SCA 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 performed to confirm the sensitivity and reliability o f 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 sensitizerfollowed was that described in: MAGNUSSON, B. and KLIGMAN, A.M. (1970) Allergic Contact Dermatitis in the Guineapig: Identification o f contact allergens, Thomas, C.C., Springfield, Illinois, U.S.A. This positive control study was conducted"'betwee_23` 'Je"and 17 July 1998 using fifteen female (nulliparous and non pregnant) albino guinea-pigs o f the Dunkm 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 o f MBT were administered: Intradermal injection: Topical application: Challenge application: 10 % w/v in Alembicol D 83.3% w/v in Alembicol D 83.3 and 40% w/v in Alembicol D RESULTS INDUCTION Intraderm al injections - Slight to well-defined irritation was seen in test animals at sites receiving MBT, 10% w/v in Alembicol D and slight irritation was observed in control animals receiving Alembicol D. 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 MBT produced evidence of skin sensitisation (delayed contact hypersensitivity) in all of the ten animals, thus confirming the sensitivity and reliability o f the experimental technique. : 24 : DPT 432/985069/SS APPENDIX 3 (continued) Individual dermal reactions after challenge application of MBT Guinea-pig number 2460 2461 2462 2463 2464 CONTROL ANIMALS E = Erythema . Score 0 = Oedema 24 Hours Ap 48 Hours Ap E 0000 0 0000 E 0000 0 o .... ----- 0---------- - 0-- 0 E 0 - - 0 0 0 -- ... - ........... 0 0000 E 0000 0 0000 E 0000 0 0000 Guinea-pig E = Erythema number O = Oedema 2465 2466 2467 2468 . 2469 2470 2471 2472 2473 2474 E O E O E O E O E O E O E O E *O E. O E O oo oo * TEST ANIMALS Score 24 Hours AP 2i 10 iI I0 48 Hours AP 02 Ml 1o ii 1* 0* . 1 1 01 01 1 0 11 1 1 01 01 1 0 10 2 2 02 NE2 2 2 2 02 2 221 2 1 1*. 0 2 1 02 01 1 0 1o 2 2 02 2 2 12 1 7 222 2 2 2* 2* Results Positive (+) Negative (-) Inconclusive () + + + + + + + + + + * Dryness and sloughing ofthe epidermis 0 Thickening, dryness and sloughing of the epidermis NE Necrotic edge A Anterior site, exposed to MBT, 83.3% w/v in Alembicol D P Posterior site, exposed to MBT, 40% w/v in Alembicol D : 25 : Company Sanitized. oes not contain TSCA CBi