Document ExpjjXEkGq6kOVy1x5x3mgZKN
SPONSOR ElfAtochemSA Cours Michelet
La Defense 10 92091 Paris-la-Defense CEDEX
France
AR226-3172
TEST SUBSTANCE
^STUIfTTITLEr SKIN SENSITIZATION TEST
IN GUINEA PIGS
(Maximization method ofMagnusson and Kligman)
STUDY DIRECTOR Xavier Manciaux
STUDY COMPLETION DATE 14 December 2000
TESTFACILITY
err
Centre International de Toxicologie BP 563 - 27005 Evreux - France
IFM Recherche SM.C.AU wn'/u. OE s ssc ooo f
LABORATORY STUDY NUMBER 20023 TSG
company Ss^of n^-"- ,,,,( .-.".-. r<"*H '""i
V_i.<uu!ys; ^ariit&sg. Sess not contain TSCA CBi
CENTRE MTERNATIONAl. DE TOXICOLOGIE
B;P. 563 27005 Evreux Cedex France
CONTENTS
STATEMENT OF THE STUDY DIRECTOR
OTHER SCIENTIST INVOLVED IN THIS STUDY
STATEMENT OP QUALITY ASSURANCE UNIT
SUMMARY
RESUME
8
1.
INTRODUCTION
10
2.
MATERIALS AND METHODS
10
2.1
TEST SUBSTANCE AND OTHER SUBSTANCES
10
2.1.1
Identification of the test substance
10
2.1.2
Vehicle
10
2.1.3
Dosage form preparation
10
2.1.4
Other substances
11
2.2
TEST SYSTEM
11
2.2.1
Animals
11
2.2.2
Environmental conditions
11
2.2.3
Food and water
12
2.3
TREATMENT
12
2.3.1
Preliminary test
12
2.3.2
Main study
13
2.3.2.1 Preparation of the animals
13
2.3.2.2 Induction phase by intradermal and cutaneous routes
13
2.3.2.2.1 mtradermal route
13
2.3.2.2.2 Cutaneous route
13
2.3.2.3 Challenge phase
14
2.4
SUMMARY DIAGRAM
15
Figure 1: Treatment sites
15
2.5
SCORING OF CUTANEOUS REACTIONS
16
2.6
CLINICAL EXAMINATIONS
16
2.7
BODY WEIGHT
16
2.8
2.8.1 2.8.2 2.8.3
PATHOLOGY
Necropsy Skin samples Microscopic examination
16
16 16 16
Sanitized. Does not contain TSCA CBS
W-i*
2.9 2.10 2.11 2.12
DETERMINATION OF THE ALLERGENICITY LEVEL CHRONOLOGY OP THE STUDY PROTOCOL ADHERENCE ARCHIVING
3.
RESULTS
3.1
3.2 3.2.1 3.2.2 3.3 3.3.1 3.3.2 3.3.3
4.
CHOICE OF THE VEHICLE
PRELIMINARY STUDY
Administration by intradennal route Application by cutaneous route
MAIN STUDY
Clinical examinations Body weight
Challenge phase - Scoring of cutaneous reactions
CONCLUSION
APPENDICES
1. Test article description and analytical certificate 2. Diet formula 3. Individual body weight values 4. Positive control to check the sensitivity ofDunkin-Hartley guinea pigs
16
17
18
18
19
19
19 19
20 20 20 20
21 22
23 24 27 29 31 and 32
.^^g^Sanlte. ^.aDorineassnnooitccontain TSCAtBt
STATEMENT OF THE STUDY DIRECTOR
dTehsecrsibtueddyinw: as performed in compliance with the principles of Good Laboratory Practice as
. (O9E8)C1D7. Principles on Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM
. Decret N 98-1312 du 31 decembre 1998 concemant les Bonnes Pratiques de Laboratoire l(lJnoduursntarlie.Officiel du ler janvier 1999), Ministere de 1'Econonaie, des Finances et de
PCroinmcmipilsessioonf GDoiroedctiLvaebo1r9a9to9r/1y IP/EraCcticoef a8s Mspaerccihfied19i9n9 CaoduanpctiilngDirteocttievcehn8i7c/a1l 8p/EroEgCresosn tthhee
hthaenPnorinnicziaptlieosnooffGlaowosd, rLeagbuolaratitoonrys Panradcatidcme iannisdtrtahteivveeprirfoicvaistiioonnsorfetlahteinirgatpoptlihceataiopnpslication of
on chemical substances (OJ No. L 77 of 23.3.1999).
for tests
tIhdeercelsaureltsthoabttathinisedredpuorritngcomnsetipteurtefosrma atrnuceeaonfdmfaeitshtufudly.record of the procedures undertaken and
FTrhainscset.udy was performed at CIT, Centre International de Toxicologie, BP 563, 27005 Evreux,
Toxicology
L&U^,
X.Manciaux ' Study Director
Doctor of Pharmacy
Date: 14 December 2000
OTHER SCIENTIST INVOLVED IN THIS STUDY
For Pharmacy:
P.O. Guillaumat
Doctor of Pharmacy
n-oi
TSCA r.y
STATEMENT OF QUALITY ASSURANCE UNIT
Type of inspections
Protocol Report
Inspections
22 March 2000 22 November 2000
Dates
Reported to Study
Director (*)
23 March 2000 7 December 2000
Reported to
Management (*)
23 March 2000 7 December 2000
m addition to the above-mentioned inspections, at about the same time as the study described in the present report, "process-based" and routine facility inspections of critical procedures relevant to this study type were also made by the Quality Assurance Unit.
The findings of these inspections were reported to the Study Director and to CTT Management. PTrhineciinpslepsecotifoGnsoowderLeabpoerrafotormryePdrianctcicoem. pliance with CIT Quality Assurance Unit procedures and
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.
^a^S0
L. Valette-TaIbi
Date: 14 December 2000
Doctor of Biochemistry
Head of Quality Assurance Unit
(*) The dates indicated correspond to the dates of signature of audit reports by Study Director
and Management. ipanr'^anifeed. Does no'ii contain T8CA 681
SUMMARY
^BBB----------fJff) At the request of Elf Atochem SA, Paris-la-Defense, France, the potential of the test substance
guinea
pigs
according
to
the
to induce delayed
maximization method of
contact hypersensitivity was Magnusson and Kligman
evaluated
in
(No. 406,17th July 1992) and EC (96/54/EEC. B.6.30 July 1996) guidelines.
and to OECD
RThegeulsattuiodnys.was conducted in compliance with the principles of Good Laboratory Practice
Methods
aTnhdiratytrgeuaitneedagproigusp woefrteenalmloaclaetsedantdo ttewnofegmroaulepss.: a control group of five males and five females
aOnnimdaalys: 1, three pairs of intradermal injections were performed in the interscapular region of all . Freund's complete adjuvant (PCA) diluted at 50% (v/v) with 0.9% NaCI (both groups),
. atelostnesu(cbostnatnrocel gartouthpe), chosen concentration in the chosen vehicle (treated group) or vehicle
. test substance at the chosen concentration in a mixture FCA/0.9% NaCI 50/50 (treated group) or vehicle at the concentration of 50% (w/v) in a mixture PCA/0.9% NaCI 50/50 (control
group).
(O1n0%d,ayw/7w, )thine osradmere toreignidouncreelcoeciavledirraitattoiopnic.al application of sodium lauryl sulfate in vaseline
On day 8, the test substance (treated group) or the vehicle (control group) was applied topically to the same test site, which was then covered by an occlusive dressing for 48 hours.
On day 22, application
all
animals
of
the
treated
and
control
groups
were
challenged
by
a
cutaneous
the
vehicle
of me test substance to the right flank.
only. Test substance and vehicle
The
left
flank
served
as
control
and
received
24 hours.
were maintained under an occlusive dressing for
Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing.
Test substance concentrations were as follows:
Induction (treated group)
,, _ . intradermal injections (day I): isotonic saline solution (0.9% NaCI), . topical application (day 8):'"
at the concentration of 5% (w/w) in sterile
undiluted.
Challenge (all groups) . topical application (day 22):
'undiluted.
At the end of the study, animals were killed without examination of internal organs. NSkoinhissatomlopgleiscawl eerxeatmakineantifornomwatshepcehrfaolrlemnegde. application sites of all the animals of treated group.
Results
No clinical signs and no deaths were noted during the study. cAofntetrrolthgerocuhpa. llenge application, no cutaneous reactions were observed in the animals of the In the treated group, cutaneous reactions attributable to delayed contact hypersensitivity(discrete or moderate erythema, sometimes associated with oedema) were observed in all animals.
Conclusion
Under our experimental conditions and according to the maximization method of Magnusson hypersensitivity in 20/20 (100%) guinea pigs.
According to the classification criteria laid down in Directive 93/21/EEC (27ffi April 1993) adapting to technical progress for the eighteenth time Council Directive 67/548/EEC,
substance should be considered as a skin sensitizer.
the test
IgSip^ySartlized.Ooes nol contain TSCA CW
RESUME
A la demande de Elf Atochem i--------------^
SA,
Paris-la-Defense,
France,
Ie
potentiel
du
produit^f--Bl
a induire une hypersensibilisation cutanee retardee est evalue chez Ie
Cobaye selon la lignes directrices
methode de maximisation de 1'OCDE (n0
de
Magnusson
et
Kligman
et
confonnement
aux
1996).
406, 17 juillet 1992) et de la CEE (96/54/EEC, B.6, 30 juillet
L'etude est realisee confonnement aux regles de Bonnes Pratiques de Laboratoire.
Methodes
gTrroeunptee ctroabitaeyedse s1o0nmt arleepsarettis10enfem2 eglrleosu.pes : un groupe temoin de 5 males et 5 femelles et un
iAriuterjsocuarpul1a,ire3 depatoiruess leds'iannjeicmtiaounxs : intradermiques sont effectuees au niveau de la region
. Adjuvant complet de Freund (FCA) dilue a 50 % (v/v) dans du NaCI a 0,9 % (groupe traite et groupe temoin),
. p(grrooduupiet atemteostienr),a la concentration choisie dans Ie vehicule (groupe traite) ou vehicule seui
.
produit a traite)
tester a
la concentration choisie dans
une
mixture FCA/ NaCI
a
0,9
%
50/50
(groupe
ou vehicule a la concentration de 50 % (p/v) dans une mixture FCA/ NaCI a 0,9 %
50/50 (groupe temoin).
Au jour 7, une application cutanee de laurylsulfate sodique a 10 % (p/p) dans de la vaseline est effectuee sur la meme zone dans Ie but d'induire une irritation locale. sAiute,joquuri e8s, tIeenpsruoitdeuriet c(goruovueprtedt'ruanitpe)anosuemIeevnet hoicccullues(igf rpoeunpdeantet m48oihne)usreosn.t appliques sur Ie meme Au jour 22, tous les animaux des groupes traite et temoin recoivent une application cutanee declenchante de produit sur Ie flanc droit. Le flanc gauche sert de temoin et recoit Ie vehicule seul. Le produit et le vehicule sont maintenus sous pansement occlusif pendant 24 heures. devaluation des reactions cutanees est effectuee environ 24 et 48 heures apres 1'enlevement du
pansement.
Les concentrations de produit sont les suivantes :
Induction (groupe traite)
. NinajeCcItiaon0s,9in%tr,adermiques (jour 1) :^--------------a la concentration de 5 % (p/p) dans du
. application cutanee (jour 8) : ttB----H_B_P_1_1011 dilue.
Application declenchante (tous les groupes)
. application cutanee (jour 22): fB----------Bnon dilue.
A la fin de 1'etude, les animaux sont sacrifies sans examen des organes internes. lDeessapnrimelaeuvxemtreanittessc. utanes sont effectues au niveau des sites d'application declenchante chez tous Aucun examen histologique n'est realise.
^MtpaalySaumzea. Ooes ftot eonfa?n TSCA C8r
Resultats
Aucun signe clinique ni aucune mortalite ne sont notes pendant 1'etude. gArporuepse 1t'eamppoliinc.ation declenchante, aucune reaction cutanee n'est observee chez les animaux du Dans Ie groupe traite, des reactions cutanees attribuables a une hypersensibilisation arentiamrdaeuex.(erytheme discret ou modere, parfois associe a un oedeme) sont notees chez tcouutsanleese
Conclusion
Dans nos conditions experimentales et selon la methode de maximisation de Magnusson
Kligman,
Ie
produittf------i----MIMMIW)
induit
des
reactions
cutanees
et attribuables
une hypersensibilisation cutanee retardee chez 20/20 (100 %) Cobayes.
a
Selon les criteres de classification decrits dans la Directive 93/21/CEE (27 avril 1993)
dix-huitieme
adaptation
au
progres
technique
de
la
Directive
67/548/CEE,
Ie
portant produit est
considere sensibilisant par contact avec la peau.
'jSSmpafyan!Szffeo9w.nofeonfa&i fSCA CBT
1. INTRODUCTION
f H H | U | | The objective of this study, performed according to the maximization method ofMagnusson and
KUgman (1), was to evaluate the potential of the test substance delayed contact hypersensitivity in guinea pigs.
to induce
mThaeterreiasluilntshoufmtahness. tudy are of value in predicting the contact sensidzation potential of the test
The study was conducted in compliance with: . OECD guideline No. 406,17th July 1992, . EC Directive No. 96/54/EEC, B.6, 30 July 1996.
2. MATERIALS AND METHODS 2.1 TEST SUBSTANCE AND OTHER SUBSTANCES
2.1.1 Identification of the test substance The test substance )^--------------1used in the study was supplied by the Sponsor.
It was identified as follows: . name:
batch number:
____ Elf Atochem filing number^l----|
description: brown liquid
. container: one plastic flask
. date of receipt: 24 March 2000 . storage conditions: at room temperature and protected from light
. composition: see analytical certificate
. expiry date: November 2000.
Data relating description
to
the
characterisation
of the
test
substance
are
documented
in
a
test article
and an analytical certificate (presented in appendix 1) provided by the Sponsor.
2.1.2 Vehicle
The choice of the vehicle was based on tests to check the homogeneity (visual check) of the preparation (for cutaneous application and intradermal injections) and its free passage through a cnaeleleddlet(hfeormianxtriamdaelrmpraalcitnicjeacbtlieoncso)n.cTehnterahtiigohness. t concentrations which satisfied these criteria were
9T2h3e16veSheicvrlees,usFerdanwcea)s. 0.9% NaCI, batch Nos. LR92803 and 2934/1 (Laboratoire Fresenius,
2.1.3 Dosage form preparation
All dosage form preparations were made freshly, on the morning of administration and any
unused material was discarded that same day.
(1) Magnusson B. and Kligman A.M.: The identification of contact allergens by animal assay. The guinea pig maximization test. J. Invest. Derm., 52: 268-276 (1969).
2.1.4 Other substances The other substances used were Freund's complete adjuvant, batch Nos. 79H8938 and 119H8927 (Sigma, 38297 Saint-Quentin-Fallavier, France); sodium lauryl sulfate, batch No. 107H0006 P(Shiagrmmaa,ce3u8ti2q9u7e FSraainnct-aQisuee, n7t7in0-0F0alMlaevliuenr,, FFrraannccee)). and vaseline, batch No. 1572 (Cooperative
2.2 TEST SYSTEM
2.2.1 Animals
Species and sex: male and female guinea pigs.
Strain and sanitary status: Hartley Cri: (HA) BR, Caesarian obtained, Antibody Free (COBS- VAF)
Barrier sustained -
Virus
Reason for this choice: species generally accepted by regulatory authorities for this type of study. The strain used has been shown to produce a satisfactory sensitization response using known
sensitizers.
Breeder: Charles River France, 76410 Saint-Aubin-les-Elbeuf, France. Number: . one male and three females for the preliminary test,
. 30 animals (15 males and 15 females) for the main test. Females were nulliparous and non-pregnant. Allocation of the animals to the groups: on day -1, the animals were weighed and randomly allocated to two groups: a control group of ten animals (five males and five females) and a treated group of 20 animals (ten males and ten females). Age/weight: on day 1, the animals of the main test were 1-3 months old and had a mean body weight standard deviation of 367 22 g for the males and 363 16 g for the females. Acclimation: at least 2 days before the beginning of the study. Identification of the animals: ear-tattoo.
2.2.2 Environmental conditions
The conditions in the animal room were set as follows:
. temperature: 21 2C
. relative humidity: 30 to 70% . light/dark cycle: 12 h/12 h
. ventilation: approximately 12 cycles/hour of filtered, non-recycled air.
The temperature and relative humidity were under continuous control and recording. The records were checked daily and filed. In addition to these daily checks, the housing conditions and
corresponding instrumentation and equipment are verified and calibrated at regular intervals.
During the acclimation period and throughout the study, the animals were housed individually in
polycarbonate cages (48 cm x 27 cm x 20 cm) equipped with a polypropylene bottle.
.
Dust-free sawdust was provided as litter (SICSA, 94142 Alfortville, France).
Bacteriological and chemical analyses of the sawdust, including the detection of possible
contaminants (pesticides, heavy metals), are performed regularly by external laboratories.
The results of these analyses are archived at CIT.
r ^anited. Does not contain TSCA C8P
2.2.3 Food and water
During the study, the animals had free access to "106 pelleted diet" (UAR, 91360 Villemoisson-
sur-Orge, France). Pood is analysed regularly by the supplier for composition and contaminant levels. The diet formula is presented in appendix 2.
Drinldng.water filtered by a FG Millipore membrane (0.22 micron) was provided adiibitum. Bacteriological and chemical analyses of the water and diet, including the detection of possible lcaobnotraamtoinriaenst.s (pesticides, heavy metals and nitrosamines), are performed regularly by external The results of these analyses are archived at CIT.
No contaminants were known to have been present in the diet, drinking water or bedding
material at the study.
levels
which
may
be
expected
to
have
interfered
with
or
prejudiced
the
outcome
of
2.3 TREATMENT
2.3.1 Preliminary test mA apinreslitmudiyn.ary test was conducted in order to determine the concentrations to be tested in the
By intradermal route (tested concentrations: 75%, 50%, 25%, 10%, 5% and 1% (w/w)): . 24 hours before treatment, the dorsal region of the animals was clipped, . iinntterarsdcearpmuallarardemgiinoins,trations of the dosage form preparations (0.1 ml) were performed in the . icnujteacnteioonuss. reactions were evaluated approximately 24, 48 hours and 6 days after the
By cutaneous route (tested concentrations: 100% and 50% (w/w)):
. 24 hours before treatment, both flank regions of the animals were clipped,
. the filter paper of a chamber (Finn Chamber) was fully-loaded with the dosage form preparations. The chamber was then applied to the clipped area of the skin (one concentration
per flank). The chamber was held in place by means of an occlusive dressing for 24 hours,
.
cutaneous dressings.
reactions
were
evaluated
approximately
24
and
48
hours
after
removal
of
the
Criteria for selectidh of concentrations The following criteria were used: . the concentrations should be well-tolerated systemically and locally, . intradermal injections should cause moderate irritant effects (no necrosis or ulceration of the
skin),
. cutaneous application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration,
. cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effects.
ll^oes^.^CQn^aw'ye^"' '"'>
2.3.2 Main study
2.3.2.1 Preparation of the animals
For all animals, the application sites were: . clipped on days -1 and 7 (interscapular region 4 cm x 2 cm), . clipped and shaved on day 21 (each flank 2 cm x 2 cm), . clipped on day 25, before skin sampling, for the concerned animals (each flank 2 cm x 2 cm).
2.3.2.2 Induction phase by intradermal and cutaneous routes
2.3.2.2.1 mtradennal route On day 1, six injections were made deep into the dermis of a 4 cm x 2 cm clipped interscapular area, using a needle (diameter: 0.50 x 16mm) mounted on a 1 ml plastic syringe (0.01 ml graduations).
Three injections of 0.1 ml were made into each side of this interscapular region (i.e. three pairs of
sites), as follows:
Injection
Site Anterior
Treated group
FCA at 50% (v/v) in 0.9% NaCI
Control group
FCA at 50% (v/v) in 0.9% NaCI
Middle
test substance at 5% (w/w) in 0.9% NaCI
0.9% NaCI
Posterior*
test substance at 5% (w/w) in a mixture PCA /0.9% NaCI 50/50
vehicle at 50% (w/v) in a mixture PCA/0.9% NaCI 50/50
FCA:
*
:
Preund's complete adjuvant
The test substance was first dissolved in the aqueous phase prior to mixing with PCA. The final concentration of the test substance was equal to that used in injection 2.
The anterior and middle pairs of injections were performed close to each other and nearest the head, while the posterior pair was performed towards the caudal part of the test area.
2.3.2.2.2 Cutaneous route On day 7, the interscapular area was clipped. As the test substance was shown to be non-irritant during the preliminary test, the animals were
treated with 0.5 ml of sodium lauryl sulfate at the-concentration of 10% (w/w) in vaseline, in
order to induce local irritation.
On day 8, a pad of filter paper (approximately 8 cm2) was fully-loaded with the undiluted test substance and was then applied to the interscapular .region of the animals of the treated group. The animals of the control group received an application of the vehicle alone under the same experimental conditions.
The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster.
SanBbea Hoes fioi e-oniate TSCA CBI
2.3.2.3 Challenge phase
On day 22, the animals of treated and control groups received an application of the test substance and vehicle. The filter paper of a chamber (Finn Chamber) was fully-loaded with the undiluted test substance and was then applied to a clipped area of the skin of the posterior right flank of all animals. The vehicle was applied under the same experimental conditions to the skin of the posterior left flank.
Ssmpany Sanitized, Does no! contain TSCA CW
2.4 SUMMARY DIAGRAM
Figure 1: Treatment sites
Indiiction-site Ihtradermal injections (day 1)* Cutaneous application (day 7): Sodium lauryl sulfate 10% in vaseline Cutaneous application (day 8): vehicle (control group) or test substance at the chosen concentration (treated group) Challenge application sites Cutaneous application (day 22)
* Intradermal injections:
(1) 50% Freund's complete adjuvant and 0.9% NaCI @ vehicle (control group) or test substance at the chosen concentration in the vehicle
(treated group) vehicle at 50% (control group) or test substance at the chosen concentration (treated group) in the mixture Freund's complete adjuvant/0.9% NaCI (50/50)
2.5 SCORING OF CUTANEOUS REACTIONS
Twenty-four and 48 hours after removal of the dressing of the challenge application, both flanks of the treated and control animals were observed in order to evaluate cutaneous reactions, according to the following scale:
. no visible change.................................................................................................................. 0 . discrete or patchy erythema.................................................................................................. 1 . moderate and confluent erythema......................................................................................... 2 . intense erythema................................................................................................................... 3
Any observed oedema was recorded. Any other lesions were noted.
2.6 CLINICAL EXAMINATIONS
The animals were observed at least once a day during the study in order to check for clinical signs and mortality.
2.7 BODY WEIGHT
The animals were weighed individually on the day of allocation into the groups, on the first day of the study (day 1) and on the last day of the study (day 25).
2.8 PATHOLOGY
2.8.1 Necropsy
At the end of the study, all the animals were killed by carbon dioxide asphyxiation. No necropsy was performed.
2.8.2 Skin samples At the end of the study, skin samples were taken from the posterior left and right flanks of all the animals showing skin reactions (all the animals of treated group). The samples were preserved in 10% buffered formalin.
2.8.3 Microscopic examination No histological examination was performed.
2.9 DETERMINATION OF THE ALLERGENICITY LEVEL
The animals of the treated group show a positive reaction if macroscopic cutaneous reactions are
clearly visible (score >. I) and are of greater intensity and/or duration of response than the
maximum reaction seen in control animals, or if macroscopic reactions are confirmed at
microscopic examination as being due to the sensitization process.
Determination of the allergenicity level The allergenicity level of the test substance is calculated by comparing the number of animals showing positive reactions with the number of surviving treated animals at the end of the study.
^^^aHv Sfflilibed. Does notcontato TSCA CS?'
'
'
"^BBi,*-;,"* - -----.--------.
% of animals
showing a reaction 0- 8 9 - 28
29 - 64 65 - 80 81 - 100
Allergenicity level
I n m
IV
V
Classification
weak mild moderate strong extreme
According to the Commission Directive 93/2 I/EEC, when the reactions are positive in at least 30% of the treated animals, the test substance has sensitization properties and the symbol Xi, the indication of danger "Irritant" and the sentence "R 43: May cause sensitization by skin contact" must be applied.
The sensitivity of the experimental technique is regularly assessed using a known moderate sensitizer, MERCAPTOBEiNZOTHIAZOLE. In a recent study performed under CIT experimental conditions, the strain of guinea pigs used showed a satisfactory sensitization
response in 80% animals (see appendix 4).
2.10 CHRONOLOGY OF THE STUDY
The chronology of the main test is summarized as follows:
Procedure
Arrival of the animals Weighing and allocation of the animals into groups
Weighing, induction by intradermal injection Sodium lauryl sulfate application Induction by cutaneous route Removal of occlusive dressings Challenge cutaneous application Removal of occlusive dressings
Scoring of cutaneous reactions after .24 hours
. 48 hours
Weighing, sacrifice of the animals and skin samples
Date
27 April and 3 May 2000 4 May 2000 5 May 2000
11 May 2000 12 May 2000 14 May 2000 26 May 2000 27 May 2000
Day -9 and -2
-1 1 7 8
10 22 23
28 May 2000
24
29 May 2000
25
29 May 2000
25
^^-"--"^
2.11 PROTOCOL ADHERENCE
The study was performed in accordance with the Study Protocol No.^fBHRand subsequent
amendments, with the following deviation from the agreed Study Protocol: the acclimation period was reduced to 2 days for four animals of the control group. This minor deviation was not considered to have compromised the validity or integrity of the
study.
2.12 ARCHIVING The study documentation and specimens generated during the course of the study are archived at CIT, 27005 Evreux, France, for 10 years after the end of the in vivo phase of the study.
The archived study materials include: . protocol and possible amendments, . raw data, . correspondence, . filial report and possible amendments, . histological specimens:
- tissues in preservative.
On completion of this period, me archived study materials will be returned to the Sponsor, or may be archived at CIT for a further period. Hi addition, raw data not specific to the study including, but not limited to, certificates of analyses for food, water and bedding (if applicable) and records of environmental data and equipment calibration, are also archived at CIT and retained for at least 30 years.
f^s.^^1*'""1''"^0'"
3. RESULTS
3.1 CHOICE OF THE VEHICLE
The vehicle chosen was 0.9% NaCI: a homogeneous dosage form preparation was obtained whatever the proportion. The dosage form preparation at the concentration of 75% (w/w) passed freely through a needle and into the dermis.
3.2 PRELIMINARY STUDY
3.2.1 Administration by intradermal route Results were as follows:
Animal number
Concentration of the test substance % (w/w)
24 hours
Scoring after treatment 48 hours
6 days
male 301
75+FCA
N
N
-
75
NN -
50+FCA
N
N
-
50
NN -
25 + FCA
N
N
-
25
NN -
female 302
75+FCA
N
N
-
75
NN -
50+FCA
N
N
-
50
NN -
25 + PCA
N
N
-
25
NN -
female 303
10 + FCA
I
10
I
N
A
N
A
5+FCA
I
I
I
5
LJ
LI
LI
1+FCA
I
I
I
1
U
LI
LI
female 304
10 + FCA
I
I
A
10
I
N
A
5+FCA
I
I
I
5
LI
U
LI
1+FCA
I
I
I
1
LI
LI
LI
FCA: mixture Freund's Complete Adjuvan/0.9% NaCI 50/50 (v/v)
N : necrosis
I
: irritation
LI : slight irritation
^^^e--.-----------'"^'-"
A
: crusts
: not performed
In order to respect the criteria for the selection of concentrations (the concentration should be well-tolerated systemically and locally, intradermal injections should cause moderate irritant effect but no necrosis or ulceration of the skin), concentration chosen for the main study was 5% (w/w).
3,2.2 Application by cutaneous route Results were as follows:
Animal number
Concentration of the
test substance %
male 301
100
RF
50 (w/w)
LF
female 302
100
RF
50 (w/w)
LF
RF: right flank LP: left flank
Scoring after removal of the dressing
24 hours
48 hours
0
0
0
0
0
0
0
0
On removal of the dressing, no residual test substance was observed.
In order to respect the criteria for the selection of concentrations (die concentrations should be well-tolerated systemically and locally, cutaneous application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration, cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effect), concentration chosen for the topical application of the induction phase (day 8) and for the challenge application (day 22) was 100%.
3.3 MAIN STUDY
3.3.1 Clinical examinations No clinical signs and no deaths were observed during the study.
3.3.2 Body weight
The body weight gain of the treated animals was similar to that of the control animals
(appendix 3).
i. Does ttol contain TSCA CW
3.3.3 Challenge phase - Scoring of cutaneous reactions On removal of the dressing, no residual test substance was observed.
Scoring of skin reactions was as follows:
Sex Male
" Animal number
216 217 218 219 220
Control group
24 hours
LF
RF
48 hours
LF
RP
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Female
231
0
0
0
0
232
0
0
0
0
233
0
0
0
0
234
0
0
0
0
235
0
0
0
0
Sex Male
Animal number
221 222 223 224 225 226 227 228 229 230
Treated group
24 hours
LF
RF
48 hours
LF
RF
0
2/Oe
0
2/Oe/S
0
2/Oe
0
2/Oe/S
0
1
0
1
0
2/Oe
0
2/Oe/S
0
2/Oe
0
2/Oe/S
0
1
0
2/S
0
1
0
1
0
2/Oe
0
1/S
0
2/Oe
0
2/Oe/S
0
2/Oe
0
2/Oe/S
Female
236
0
237
0
238
0
239
0
240
0
241
0
242
0
243
0
244
0
245
0
2/Oe 2/Oe
1 1 1
2/Oe 2/Oe
1 2/Oe
2/Oe
LF : left flank (vehicle) RF : right flank (undiluted test substance)
Oe : oedema S : dryness of the skin
LS : scoring masked by a marked dryness of the skin
0
2/Oe/S
0
1/S
0
1/S
0
1/S
0
1/S
0
2/Oe/S
0
2/Oe/S
0
2/Oe/S
0
1/S
0
LS
.S^fpajiy ]Ss8iSVSseaD.ees ftol eonlain TSC^
No cutaneous reactions were observed in the animals of the control group. In the treated group, a discrete or moderate erythema (grade 1 or 2) was noted in all animals at the 24 and 48-hour readings. An oedema was recorded in 14/20 animals. Dryness of the skin was observed in almost all animals at the 48-hour reading. The observed cutaneous reactions were attributed to delayed contact hypersensitivity. 4. CONCLUSION Under our experimental conditions and according to the maximization method of Magnusson hypersensitivity in 20/20 (100%) guinea pigs. According to the classification criteria laid down in Directive 93/2 I/EEC (27th April 1993) adapting to technical progress for the eighteenth time Council Directive 67/548/EEC, the test
substance should be considered as a skin sensitizer.
.%aq%ySfiWzeaD; oes TToleonlain T8CA CBt
APPENDICES %mpany SanWzea. Does ffol contain TSCA C8f
1. Test article description and analytical certificate Does not contain TSCA CBt
TOXICOLOGY DEPARTMENT CONFIDENTIAL
elf atochem s.a, La defense 10, Cours Michelet
DT1 733 14th March 2000
92091 Paris-la-Defense cedex, France
TEST ARTICLE DESCRIPTION
E
IDENTITY
Test article name Origin Batch TElfAtochem filing number
: Elf Atochem Villers-Saint-Paul ' fl^^^^^^^^^^^f
PHYSICAL AND CHEMICAL PROPERTIES
Appearance Specific gravity Boiling point
Flash point Solubility
Brown liquid 1070 kg/m3 at 20C (liquid) 95-C
No flash point in the test conditions: (10 - 100C) Soluble in : Alcohols, Hydroxylated solvents. Insoluble in : Hydrocarbons, DMSO
TOXICOLOGICAL INFORMATION AND USE SAFETY See Material and Safety Data Sheet
STORAGE AND DISPOSAL
Storage Expiry date Disposal
in dark and at room temperature November 2000
incineration
SairaiSfce'dti.oes net contain TSCA <
ISeffipany
elf atochem
M f f~\l----r--\
Villers Saint Paul, Ie 26 Novembre 1999
CERTIFICAT D'ANALYSE
LotN"
Extrait Sec
Ethanol
pH
Tension supcrncielle
Densitc
Foisonnement
0,5% dans cau de ville
Drainage
0,5%
dans eau de ville Pouvoir lihnogene Sur heptane / eau
de ville
Unite
% %
Mn/m
Minute
Sec.
Resultat 27,3
Specification de
fabrication
27 a 29
Methode d* analyse
LCU622
1,1
Oa2,5
LCU 604
8.6
8a9
LCU642
15,9
0 a 16,5
LCU 663
1,083
1,06 a 1,09 LCU662
9
6 a 50
LCU 670
6
3 a 50
LCU 670
14
9 a 40
LCU 668
Le Chef de Service du Laboratoire SIGNATURE:
NOM : Robert Gruppo
^llteed.^8"01
GW^
2. Diet formula
.^noleonlalnTSCACW
Ref: 106
COMPLETE DIET GUINEA PIG MAINTENANCE DIET
Appearance: 4.5 mm diameter granules Conditioning: bags of 25 kgs
Daily portion: Guinea pigs 35-50 g, water adiibitwn.
FORMULA %
Cereals ..................................... Grain byproducts and legumes. Vegetable protein (soya bean meal, yeast) .............................. Vitamin and mineral mixture...,
AVERAGE ANALYSIS %
Calorific value (Kcal/kg). Moisture........................... Proteins............................ Lipids............................... Carbohydrates (N.F.E.) Fibre ................................. Minerals (ash) ..................
AMINO ACID VALUES
(calculated in mg/kg)
Arginine..... Cystine....... Lysine......... Methionine. Tryptophan. Glycine.......
FATTY ACID VALUES
(calculated in mg/kg)
Palmitic acid ...... Palmitoleic acid. Stearic acid......... Oleic acid........... Linoleic acid ...... Linolenic acid....
42 46
9 3
2600 10 17
3 49 13
MINJERALS (calculated in mg/k^g)
Nat.
CMV
P................... Ca ............... K
................. Na ...............
Me Mn ...............
Fe................. Cu ................ Zn ................ Co ................ I.................... Cl
.................
val.
7400 5400 12000 1300
3270 60
170 10 ^4\0/
0.1 0
0
val. 1jt4-t0W0
5600
0
1950 130 4~T0\J 150 15 45
1.5 0
0
Total oBooUnUn 11000
niZnu/uvui
Tj^pijs^nj ^J~4TnUnU
100
320
2^5^ 6oJ< 1.6
0
0
8
8500 2500 7200 2100 2000 6000
3600 0
700 5900 11200 3000
vnrAMDSTS (calculated per kg)
Nat.
CMV
Vitamin A Vitamin D3
Vitamin Bl Vitamin B2
Vitamin B3 Vitamin B6 Vitamin B12 Vitamin C Vitamin E Vitamin K3 Vitamin PP
-
Polic acid
P.A.B. acid Biotin
Choline
Meso-mositol
val.
3500 IU 30 IU
6mg 5mg
22 mg
0.7 mg 0.003 mg
Omg 15 mg 5mg 97 mg
2.2 mg Omg
0.02 mg 1010 mg
Omg
val.
7500 IU 2000 IU
6.4 mg
6.4 mg 26 mg
2.7 mg 0.012 mg
400 mg 60 mg
12.6 mg 14.5 mg
1.3mg
2.5 mg 0.06 mg
60 mg
62.5 mg
Total
11000IU 2030 IU
12.4mg
11.4mg 48 mg
3.4mg 0.015 mg
400 mg 75 mg
17.6 mg 111.5mg
3.5 mg
2.5 mg 0.08 mg 1070 mg 62.5 mg
This food is supplemented with stabilized coated vitamin C, avoiding the need of other food substances (greenery, ascorbic acid) if used within 4 months of date of manufacture.
UAR, 7 rue Gallieni, 91360 Villemoisson - Tel: 01.69.04.03.57 - Fax : 01.69.04.81.97 (Ref. Doc. UAR : 1992)
^SSSBSSSSaslHzecI. Uses fibt contain TSCA CBf
^"----~L-,". *'- '.
3. Individual body weight values
Groups Sex
1
Male
Female
2
Male
Female
ENDIVID UAL BODY WEIGHT VALUES (g)
Days
Animals -1
1
(1)
25
216
334
334
192
526
217
340
347
146
493
218
358
368
156
524
219
300
313
147
460
220
375
392
99
491
M
341
351
148
499
SD
28
30
33
27
231
385
391
90
481
232
344
359
120
479
233
353
370
63
433
234
354
379
122
501
235
360
369
111
480
M
359
374
101
475
SD
16
12
25
25
221
367
367
84
451
222
352
364
135
499
223
351
366
177
543
224
349
357
160
517
225
377
386
120
506
226
379
388
138
526
227
364
373
187
560
228
368
374
170
544
229
392
392
122
514
230
377
381
157
538
M
368
375
145
520
SD
14
12
31
31
236
352
358
26
384
237
353
367
144
511
238
371
376
97
473
239
330
337
115
452
'
240
337
341
49
390
241
350
354
85
439
242
331
338
113
451
243
350
360
76
436
244
360
376
128
504
245
352
366'
131
497
M
349
357
96
454
SD
13
15
38
44
(1) = Body weight gain
M =Mean
SD = Standard Deviation
iffol contain TSCACBI
4. Positive control to check the sensitivity ofDunkin-Hartley guinea pigs
^s^.------------------8'
Purpose: check the sensitivity of Dunkin-Hartley Guinea pigs (Breeder: Charles River France) to a positive control test article
Method
Magnusson and Kligman
Test substance
Crr Study-Date
MERCAPTOBENZOTHIAZOLE
CIT/Study N^M------i------
Number of animals
one control group of 5 animals and one treated group of 10 animals
induction
l%(w/w)
20% (w/w)
intradennal route day 1 cutaneous route day 8
Challenge application: 20% (w/w)
cutaneous route day 22
Conclusion
Under our experimental conditions and according to the Magnusson and Kligman method, the test substance MERCAPTOBENZOTHIAZOLE at the concentration of 20% (w/w) induced positive skin sensitization reactions in 80% guinea pigs.
INDIVIDUAL REACTIONS: CHALLENGE PHASE MACROSCOPIC FINDINGS
Groups Sex
Animals
24-hour
LF
RF
48-ho ur
LF
RF
Control Female
16
17
18
19
20
0
0/C
0
0/S/C
0
0/C
0
0/S/C
0
0/C
0
0/S/C
0
0/C
0
0/S/C
0
0/C
0
0/S/C
Treated Female
21
22
23
24
25
26
27
28
29
30
0
2/C
1/S
2/C/S
0
2/C
0
2/C/S
0
2/C
0
2/C/S
0
3/C
0
LS/C
0
1/C
0
0/S
0
2/C
0
LS/C
0
3/C
0
LS/C
0
1/C
0
LS/C
0
2/C
0
LS/C
0
2/C
0
0/S/C
LF left flank (vehicle)
RF right flanJc (test substance at the concentration of 20% (w/w))
S
dryness of the skin
LS scoring masked by dryness o f the skin
C
yellow coloration of the skin
negative
+
hypersensitizing reactions
Conclusion
-
+ + + +
-
+ +
-
+ +
stconlalnTSCACBI
