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l . Signatures

Study Director

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Management.- 

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2 . Summary This study was ed to assess tential irritant / corrosive effects of 'ty: 95. n theskin ofrabbits. The results are summarized in the table below . Table 2-1 Summary of Intent Effects (Score)

. Oedema formation 0 0 0 0.0 . - no 3 and eschar brtnation 0 0 0 0 .0 - no . ryt ema (redness) 2 and eschar formation 0 0 0 0.0 - no Oedema formation . 0 0 0 0.0 - no ryt ema (redness) "mema (reoness) and eschar iQrmation 0 0 0 0.0 - no Oedema brtnation 0 0 0 0.0 - no . Mean . Reversible Animal 24 h 48 h 72 h scams Response (days)

no posidwrosponse : msan scores < 2 poNtive response : mean wore* t Z no : not epplicable ' s 1 In all animals the slightly bladc discoloratlon of the treated skin by test compound was reversible within 1 day respectively 2days. ' . According to~classi8cation crlte not an lrritant to the skin.' Conclusion:

There were nosystemic intolerance reaction: . The method used complied with the OECD - Guideline for Testing ofChemicals No . 404 -" Acute Dermal Irritation/Corrosion" ; adopted: 24th April 2002 ; EEC Directive 67/548 Annex V - Method H .4. (1967) in its currentversion.

# , PUBLIC CopyStudy initiation date : 2006-09-14 (YYYY-MM-DD) Experimental starting date : 2006-09-19

, (YYYY-MM-DD) Experimental completion date : 2006-09-22 (YYYY-MM-DD) Studycompletion date : see signature page The study objective was to determine the irritant / corrosive effects on akin of albino rabbits. Information derived fromthis test serves to indicate the possible existence ofhazards likely to arise fromshort-tamexposurE ofakin to the test substance, and - with respect to a proper handling and use - serves to permit classification . ~(labelling) ofa product. The st~~w~ n1an- raw

data and the final report are retained in the archives specified by

e storage ofa retention sample is in the

. . responsibility o ie sponsor.8



### 4 . IdentlficAtion and Responsibili 4 .1 Identification of the Stn Study No. 4 .2 Responsibilities Study Director ' .Head ofTest Facility ' Head of Department Archiving Head ofQuality Assurance Unit 

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5 . Material and Methods 

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The method used complied with the OECD- Guideline for Testing ofChemicals No . 404-" Acute Dermal Irritation/Corrosion" ; adopted: 24th April 2002 ; EEC Directive 67/548 AnnexV- Method H .4 . (1967) in its currentversion. For reasons of animal welfare a sequential testing strategy was followed in - accordance with the current version ofthe EECDirective 67/548 and the OECD Guideline No

. 404, irrespective ofthe requirements ofother guidelines for testing for dermal irritation/corrosion in rabbits. . This testing strategy comprised a stepwise approach including the evaluation of existing human and/or animal data showing effects on the skin or mucous membranes, the performanceof a SARevaluation for sldn corrosion/irritation, measurement of pHvalue, the evaluation of data on systemic toxicity via the dermal route and the performanceof avalidated in vitro test for skin corrosion (Human 3D' Epidermal Skin Model) before in vivo testing for skin irritation/corrosion in rabbits. The in vitrotest for skin corrosion is archivedJ"~b e test compound is not corrosive to the skin.

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5 .1 Test Compound Test compound Chemical name Appearance : black, powdery to coarse-grained Storage : roomtemperature Batch no . 600900b Content oftest substance : 95.8 t used for calculations) Expiry date : 2006-12-09 (YYYY-MM-DD) Confirmation ofthe identity of the test compound was performed: ' 5 .2 Test System(Anlmals) The studywas performed in female albino rabbits (strain Crl :KBL(NZW)BR, Charles River, 88353KiBlegg, Gummy) recommended as the preferred species for this type of studies. Healthy adult albino rabbits fme of clinical signs were used. The health ofthe animals was routinely examined for the main specific pathogens by the breeder. Before the study the rabbits had been vaccinated against pasteurellosis. No treatment with antibiotics was performed prior to receipt ofthe animals, or during the acclimatization phase or study period. In some cases the animals had already

, been used in earlier studies. This has no influence to the results ofthis study.11 

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Females were nulliparous and not pregnant. The acclimatization time in the test facility was at least 5 days. Body weights at start of study: 2.1 kg -2 .9kg Age : young adult animals The animals were identified by labels on the cages stating at least study number, test compound, animal number and by tattooed number assigned by the breeder. 5.3 Housing Conditions Theanimals were housed individually in cage units Metall/Noryl by EBECO . Excrement trays below

the caps contained low

dust wood granulate bedding (J. Rettenmaier 8t Mime, 73494Rosenberg, Germany) . The wood granulate was changed at. least twice weekly. The animals were regularly transferred to clean cages. Theanimal roomhad a standardized climate: Room temperature ' : 20}3°C Air humidity : 50 :k25% Light/ Dark cycle : 12 hours rhythm . The humidity andair temperature were continuously recorded. Occasionally deviations from

the standards occurred, e .g. during cleaning of the animal room or effects ofthe weather. They did not have any apparent influence on the outcome of the study . ' .

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The animals received the standard diet "SsniffK-Z" 4mm (manufacturer: Ssniff Spezialdifiten GmbH, 59494Soest, Germany), approximately 100g per animal per day~and tap water ad libitumfrom polycarbonate bottles. The nutritive composition and the contaminant content ofthe standard diet were checked andanalyzed routinely in random samples. Nothing untoward was found . The tap waterwas ofdrinking water quality (according to the Drinking Water

. Decree in the current version) and analyzed routinely. Thewood granulate was randomly checked for contaminants at regular intervals. The results ofthese analyses have been ato

_ e available data yielded no evidence of any adverse

. effects on the aim

ofthe study

. . The animal room

was cleaned at least once a week and disinfected at least once a month . It was ensured that thediet was not contaminated, and that there was no contact ofthe cleaning or disinfecting solution with the test animals. 5 .4 Exposure Procedure . On the day before the test, the fur was shom on the right and left side fromthe dorso-lateral area ofthe trunk ofeach ofthe rabbits. Care was taken to avoid abrading theskin. Only animals with healthy and intact skin were used. 0 .5, gofthe pulverized test substance moistened with water (to ensure good contact with the skin) was applied to the skin ofthe animal under a gauze patch . Thetreated skin area was approximately 2 .5 cm by 2.5 cm

in size. The patch was placed on the dorso-lateral areas ofthe trunk ofeach animal and was held in place with nonirritating tape for theduration ofthe exposure period. Thus, access by the animal to the patch and resultant ingestion ofthe test substance was prevented.13 

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'Mar the exposure period the dressing and patchwere removed. The exposed skin area was carefully washed with water without altering the existing response, or the integrity ofthe epidermis. Thesurrounding untreated sldn served as control . Dueto a possible irritant potential ofthe test substance, in the first step only one animal was used and three test patches were applied successively to this animal, as described above. The first patchwas removed after three minutes. As no serious skin reactions were observed, the second patchwas applied and removed after one hour. At this stage theobservations indicated that with respect to animal welfare the exposure can be allowed to extend to four hours, therefore the third patch was applied and reamoved after four hours and the responses were graded one hour later. The test was completed using two additional animals, exposed for four hours. Ss Clinical Observadons and Scoring Based on most recent guidelines the dermal irritation was scored approximately at

, 1, 24, 48 and 72 hours after patch removal . If no irritation indices were observed after 72 h, the study was finished. If dermal irritation was observed, animalswere monitored usually on day 7 and 14 after patch removal until the changes had completely subsided, however for not more than 14 days after application. Thedegree oferythema/esclhar formation and oedema formation was recorded as specified by DRAIZE (see Appendix Table g_1) and any serious lesions or toxic effects other than dermal imitation were also recorded andfully described., As general criteria the body weight ofeach animal was recorded at the beginning of the study . Ifclinical findings other than dermal irritations occur they were recorded daily.p. 10

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5.6 Evaluation and Interpretation of Results The interpretation ofthe results is based on thedirective 67/548/EEC and its subsequent amendments (see Appendix Table 8-2) .

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. Results The individual findings ofthe treated s1dn areas at thevarious observation times are summarized in the tables below . Thecontrol areas did not show any abnormal findings and are not listed in the table . Table 6-1 Irritant Effects on the Skin - First Animal (Exposure 3 minutes / 1 hour) Animal I Observation Enxoewe immsd after oatch romoval 3 minuEss ryMma (redraw) and ncfiar toFnalfon 0 oedema larnaron 0 Exposure 1h 0 0 Animal 1, 1h Immediately after patch removal : test compound could not be removed

completely fromthe akin, slightly block discoloration Table 6-2 Irritant Effects on the Skin (Exposure: 4hours) Animal 1, Body Weight Z

. " kp Observation a(Esr mrtaval 1 h 24 h 48 h 72 h day 7 day 14 6 0 0 0 0 - - ' 0 0 0 0 - - Animal Z, Body

. Weight 2

.2 kg Observation (after patch romovall 1 h 24 h 48 h 72 h day 7 day 14 7rynMa, FOR") anaaONr S.on 0 0 0 0 - - 0 0 0 0 - - Animal 3, Body Weight 11 kp Observation (after pstch romovalt 1 h 24 h 48 h 72 h day 7 day 14 Iiwr~~) ~ ~ ~amaoon 0 0 0 0 - - 0-18 0 0 0 0 - -

- : no further examination Animal 1, 1h" : test compound could not be removed completely fromthe skin,

1 h - 24h' : slightly black discoloration of the treated skin Animal 2, 1 h' : test compound could not be removed completely fromthe skin,

, 1 h": slightly black discoloration of the treated skin Animal 3, 1 h" : test compound could not be removed completely fromthe skin,

1 h - 24h" : slightly black discoloration of the treated skin ' s after patch removal



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Table 6-3 Summary of Irritant Effects (Score)

. Mean Reversible Animal 24 h 48 h 72 h scores Response (days) EMhema (rodness) 1 and esdvarformation 0 . 0 0 0 .0 - no pedernefonrsMon

__ 0 0 0 0 .0 - no

, Erylhema (redness) _ 2 and esohar formaUon 0 0 0 0.0 - no padema foRnatbn 0 0 0 0.0 ~ - no Erylhema ) 3 and esd a *xmaWn 0 0 0 0.0 - Oedema formation 0 0 0 0 .0 - no no posidv~s response : mean scores < 2 = - -- positive response : mean scores Z 2 = + . no : not applicable In all animals the slightly black discoloration of the treated skin by test compound was reverslblq within 1 day respectively 2days.

### 7. Conclusion - The irritant / corrosive potential o412MIwas studied on the sldn of rabbits. The method used complied with the OECD - Guideline for Testing of Chemicals No

. 404-" Acute Dermal irritation/Corrosion" ; adopted: 24th April 2002; EECDirective .67/S48Annex V- Method B .4. (1'967) in its current version Accordingto classification crite skin. , ot an irritant to the Therewere no systemic iatoleraace reactioas.

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8 . Appendix Gradinjof Skte Reactions

Table S-1 Grading of Skin Reactlons Erythema and Eschar Formation No erythema 0 Very slight erythema (baroly perceptible) 1 Wag defined aylhema 2 Moderate to severe erythema 3 Severe erytheme (beefy redness) to escharformation preventing grading of 4 erythema Maximum possible: 4 Oedema Formation

, No oedema - 0 Very slight oedema (barely perceptible) 1 Slipht oedema (edges ofarea well deilned by definite raising) 2 Moderate oedema (raised approximately 1 mm) 3 Severe oedema ' 4 (raised more than 1 mm and extending beyond area of exposure) Maximumpossible: 4 Corrosive Im :versibie damage of the skin; namely, visible necrosis through the epidermis C and Into the dermis, toNowing the application of a test substance for up to four ' hours. Corrosive reactions we typified by ukws, bleeding, bloody scabs, and, by the end of observation at 14 days, by discolouration due to blanching of the skin, complete areas of alopecis, and scan.19 . 

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interpretation of Results Table 8-2 Interpretation of Results 

### Causes severe burns Atest material that causes severe bums to skin is a test material that, If when applied to healthy intact animal skin, results in - full thickness destruction of skin tissue as a result of up to three minutes exposure Causes burns A test material that causes bums to skin is a test material that, if when applied to healthy intact animal skin, results in - full thickness destruction of skin tissue as a result of up tofour hours exposure. ~ IrrItating to skin A test material with Irritating potential to skin is a test material that produces at least in 2 of 3tested animals a positive response of: erythema/eschar' 2 2 and/or oedema* and/or t2 inflammation persisting until the end of the observation time. * : calculated as the mean scores at the reading times 24,48 and 72 hours.p. 16



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WYl~inrkhq~i O~Andd ~IaA Yn n~bnwn T1w ~Oo~ ~nlawd 1at hd~y b Mdudd Inther~llanw wWvAd +@ ' -04 me WCw*I~P~op r~ sad b YmpWMon GmpW Elrrrrrrqdr citP.NNnw4Wi obuwweM ewa , Ad e .~~ onwwlqs du my.IdoebwietAn ww+ nbied a.ae oe m . +rrpwen nreR kc. . e.qoe*m .a npeur. b"WK aw h dWw ROWxWMdo On twt 1~0~b~6b~r oeMw "w sorr~a~dudbs 1e W .WAW MbRqm wow 0n." aK ou- o~w~aw~n~n. ~r~yb . .rcxP . ~ b~Evaluation of corrosive proper" by . using an ardficiat 3D-Skin model 

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Study Completion Date: September 12, 2006

page 1 of I8

### GLP COMPLIANCE STATEMENT' This study was conducted in compliance with the OECD Principles of Good Laboratory Practice as revised in 1997 (ENV/MC/CHEM(98)17) and with the revised German Principles of Good Laboratory Practice according to Annex I German Chemicals Act (Bundesgesetzbiatt Part 1, No. 40, issued June 27, 2002) .



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INDEX 1 . QUALITY ASSURANCE STATEMENT. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . .. . . . . .. . . . . . . . .6 Z. SIGNATURES .. . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 3 : SUMMARY. . : .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 4 . INTRODUCTION. . .. . . . . . . . . . . . . . . . . . . . . . . : . .. . . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 8 4 .1 . DeMidoo »».».»»»» . .». .. .» .».». . . .».»»»_». .»». .»»»». .». . .» .. .... ... ... . ... ... . .». .»» .. .»» .»».»»»»»».»».»S 4.2. Ais.»». . .»: . .»». . . . . . .». . . . . . . .». . . .». . . . .». . . .». . .». .»». . . . . .»» .»»». . . . .»». . . . . .. .»: . . . . . . . . . . . . . . . .». . .»»». . . . . . . . . . . .». . . . . . . . .».a 5 . STUDY IDENTIFICATION AND RESPONSIBILITIES . .. . . . . . . . . . . . . . . ., . . . ., . . , g 

### 5 .1 . Studyw

.nane.tbn »»...»»». .».»».».»»»»»»».»»».».».»»».».»»»».»».»»»»».»»»».»»»».». .».»»»». ...»» 9 5.2. Reaponiibilitier»».». .». . . .». . . . . . . . . . . . .».»»»» . . . . . . . . . . . . . .». .»». .» . . . .». .»».»»»». . . . .» .»». .». . . . .»». .». . .».» .» .». . . . . .9

. . 6 . MATERIAL AND METHODS. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 6 .1 . Materia6»»». . . . .». . .»».»». . . .». .»». .». . . .».» . . .»»». . . .».» . .»». . . . . .»»». . . . . . .».»». . . . . . . : .» . .». . .»»» .». . .» . . . . .»»». . .10 e.1 .1 . Test Item

. . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 8 .1 .2 . Bufhrod eolution and negative control . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . i l

... . .. .... ... . 6.2 . Metbods. .... .. .». . . .».»... .» .»»». . ... . .. .».».. ... . .... ... .... . . .».»... . .»»»... ... .. .».».». .»» .. .». . .... ... . . . .».. . .... .11 8.Z.1 . Reoonatructsd titsuss .. . .. . . ... . . . . . .. . . .. ... . ... ... . . ... ... . . ... ... . . . . . .. . . .. . ... ... ... . .. . ... . .. . ... . . . . ... . .. . . .. . ... . ... . . .. . ... . l l B :Z.Z. Adaptatbn to call culture condiNon~... . .. . ... . . .. . .. .. .. . . .. . ... . .. . . . . . .. . . .. . .. . ... . .. . .. . . .. ... . . . . . . . . ... . .. . . ... . . . . . ... l l 8 .2.3 . EnvironmsnW

conditions. .. . . .. .... . .. . . . . . .. . .. . .... . .. . . ... . . . . . .. . . . . . . . . ... ... . . . . . .. . .. . . .. . .. . . . . . .. . ... ... . ... . ... . . . . . . .. . . .12 8.2 .4. Too itern famulation. ... ... . . .. . .. . . ... . .. .... . ... . .. . ... . . .. .... . .. . .. . . . . . . ... . .. . .. . .. . . .. . .. . :.. ... . ... .. . . .. . . .. . . .. . . .. . . .. . . .. . .12 0 .2.5. Application ofthe led material and incubation ... . . .. . .. . ... . . . . . . . . ... ... . . . . ... ... . . .. ... ... . . . . . .. . .... . ... ... . ... .12 8 .2.8 . Drbrm(nadon ofcog viability (MTT) . .. . .. . . ... . .. . . . . . .. . . .. . ... . . . . . ... ... . .. . . . . ... . ... . . . . .: . ... . .. . ... . .. . . ... . .. . . ... .. .12 8.2 .7. Reliability Chedc. . .. . . .. . . .. . ... . . .. . .. . . .. . .. .... . . . . . .. ... . . ... . .. . . .. . .. . . . . . ... . .. . . .. . .. . . .. . .. . .. . ... . .. . .. . . ... ... . .. . . ... . .. . . .. . . .13 T. RESULTS. . .. . . . . . . . . . . . . ... .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 7.1 . 7 .2. Cytotoxic indices (viability decre s se measured by 14 Summary of results 14 8. DISCUSSION AND EVALUATION . . . . . . . . . . . . . . . . . , . . ,. . . . . . . . . . . .14 9 . REFERENCES. . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15p. 21



## 10 . AeBREVIATioNS. ................................................ .. ........., .. .. .............. .... ....... .1s 11 . 11 .1 . 11 .2 . APPENDIX. . . . . .. . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . .. . . . .. .. . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .17 Reiulb .» . . . .». . . .». .». . . . . . .»». .» .» . . .» . .». . . . . . . . . . .»».»»»». . . . . .»»». . .». .»». . . . . . . . . . . . . . . . . . . . . . .» .» .»». . . .».». . . .».». . . . .17 CertMcatloa otValWatioa .». . .» :» .». .... . ... .... . . .».. ..... .. .».. . .».». .. .». . ... ... ... .».»»»»».»». . .»... . .. .»». .»»».».1s 

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# , PUBLIC COPY Quality Assurance Statement Test Item: Study No. : In the relevant laboratory areas the equipment and standard processes used in connection with toxicological short-termstudies were audited periodically by the Quality Assurance (process- based inspections) . Audit reports have been submitted in writing to the study director and to the laboratory management . Date of Study phases Date of report to study director Inspection inspected and/or management Aug . 07,2006 STUDY PLAN Aug . 07, 2006 Aug . 23, 2006 PREPARATION/PROCESSING OF SAMPLES, RAW DATA Aug

. 23, 2006 (PROCESS BASED) Sept. O1, 2006 - Sept. 01, 2006 FIRST DRAFT Sept. 01, 2006 Sept 07,2W6 FINAL DRAFT Sept . 07, 2006

The results ofthe stu and the methods used have been correctly reported. Date: ~ (O Responsible:

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. SIGNATURES

Study Director:

Head of Molecular and Special Toxioologyy: 

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### 3. SUMMARY

This study for predicting non-specific, corrosive potentials of compounds by using reconstructed human skin (RHS) was performed in 2008. The experiment was carried out on a reconstructed human epidermis EST-1000 (CeqSystems, St. Katharinen, - - ,--- -- of topically applied skin corrosives with the test item Corrosive skin effects of substances are deflned as Irreversible damage of the skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test substance for up to four hours. In vivo corrosive reactions are typified by ulcers, bleeding, and bloody scabs. A 10096 concentration was tested on the skin/epklennal equivalents In triplets. For the determination oftime related cytotoxic effecls the incubation periods were 3 min and 60 min, respectively. By this the study was conducted in accordance with the OECD431 guideline draft as well as with an ECdraft guideline (amending Council Directive 87/548/EEC, B.40 Skin corrosion) using the test itemconcentrations and incubation periods recommended there. These tests are also related to the revised OECD404 guideline "Acute Dermal Irritation/Corrosion" . To check the reliability of this test procedure a blinded positive/negative control study is conducted at regular intervals In the lab . This reliability check Is aiways performed by two technicians, two laboratories in parallel using the same batches of the control test items. The test itemwas applied at a 10096 concentration, i .e. 25mg per Insert. (plus 501r10

.996 NaCl to moisten and ensure good contact with the akin) The MTT (Methylthiazoletetrazollum) method has determined the following values of viability after 3 min. or after 80 min. of incubation: 103 .7696 and 102.5296, respectively. Thus, the results show that no corrosive property of the test itemwas deterrnined by the assay used. 

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### 4

. INTRODUCTION Corrosion of the skin is defined as an Irreversible necrotic alteration of the tissue induced by a chemical . While the testing for corrosives usually involves the rabbit skin as a predictive in vivo test method, substances may be classified according to their corrosive potential by the determination of their cytotoxic effects on an in vitro reconstructed human epidermis. The test principle is based on the MTT assay roflecft the exposure of the epidermal equivalent to topically app There are several synonyms for artificial 3D-skin models Ilke~human skin recombinants, reconstructed human skin/epidermis, 3D human skin/epidermal equivalents, in vitro engineered skin/epidermal substitutes, artificial skirVepiderrnis. The 3D-skln used for the studies described hero are also described In different guidelines or guideline draft for the investigation of iMtatinglcorroslve properties of test compounds (cf. also relevant section) . 4.1 . Deflnitlon Human full thickness skin models and reconstituted epidermal equivalents are in vitro engineered tissue cultures that provide a three dimensional architecture which is biochemically, morphologically and functionally comparable to human epidermal tisaue/skin in vivo. In contrast to this organotypic ekin explant systems are based on ex vNio skin removed fromhuman or mouse and cultured -in toto, afterwards. According to the literature all the models are useful in screening for topically applied irritating, corrosive or photocytotoxic substances. 4.2. Alm The aimof these investigations was to test for corrosive properties of the test Itemin accordance with existiiQ International guidelines and guideline drafts.

are retained in the archives specified by he storage of a retention sample of the France itemIs in the responsibility of the 

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### 5. STUDY IDENTIFICATIONANDRESPONSIBILITIES

5.1 . Study Identification The laboratory study was carried out under labo Study number

. Experimental starting date: August 08, 2006 Experimental completion date: August 08, 2008 Study completion date see signature p Sponsor . 5 .2. Responsiblllties Head of Toxicology: Study .Director: Head of Molec

:ular and Special Toxicology: Head of Quality Assurance Unit: Archiving of study data: .-



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### 6

. MATERIAL AND METHODS The model used for this study has a functional stratumcomeum with an underlying layer of living cells as recommended by the OECD431 guideline draft, and EC draft amending Council Directive 67/548/EEC, B.40.Skin corrosion. The barrier function of the stratumcomeum

is adequate, as has been shown by the supplier. The viability of the living cells in the model must be sufficiently high to . discriminate well between the positive and negative control substances. Cell viability -was measured by the amount of MTT reduction, i .e. an OD value following exposure to the negative control substance or the test item . 6 .1 . Material 8.1 .1 . Test item Test item : Batch No. : Chemical name: Content of active Ingredients : not given by the sponsor Approval : until December 09, 2008 Physical state: solid Appearance: bladc powder Storage: at roomtemperature



# PUBLIC CO6.1 .2. Buffered solution and negative control

Physiological saline solution (0 .9 96 NaCI, 501rl) was used as negative control . 6.2. Methods The methods used for this protocol have already been described in the literature and relevant guidelines or guideline drafts. Unless Internationally recognised standardised reference values or validation tests are available, the method used here must be viewed with this in mind. 6 .2.1 . Reconstructed tissues The experiment was carried out on a reconstructed human epidermis EST-1000 (Ce1lSystems, St . Katharinen, Germany) . The tissue equivalents were shipped in 24 well *cell culture plates on Agarose supplemented with maintenance medium(Kit contents EST1000 ; CeIISystems, Cat.- No.CS-1001) . Inserts were of 0.63cm2 size. All tests were performed in triplets for each concentration and each time point (3 min or 60 min) . 6.2.2. Adaptation to cell culture conditions Inserts with EST-1000 reconstructed human epidermis (0.83cm2) were packed under sterile conditions and were shipped refrigerated on supplemented Agarose. Upon, snivel, 6well culture plates were pro-filled with 1 ml of fresh and cool maintenance medium . The reconstructed tissues were placed into the prepared cell culture plates (1 insertlvirolp and were adapted to thb .recommended tissue culture conditions (596 CO2, 37°C, max humidity} afterwards for at least 8 hours before use. In case of cultivation of the skin equivalents for more than 24 hours, a daily medium change is required by aspirating the mediumand replacing it by 1 ml new maintenance medium (37°C) for each well .-12-

~ 8 .2 .3. Environmental conditions The environmental conditions in the incubator were standardised as follows: Incubator temperature: 37 :t 2' C COz gas concentration: 5 96 Humidity: maximum Occasional deviations fromthese conditions occurred e.g. as a result of opening the incubators door, but this has no apparent effect on the course or outcome of the study. All Incubation steps were performed In a COz atmosphere incubator (Heraeus, Ostenode-Germany) . . 8 .2.4. Test itemformulation . . Test itemwas used undiluted, i .e. 10096 concentration. 8.2.5 . Application of the test material and Incubation For testing of chemical induced corrosivity the EST-1000 inserts were exposed to 25mg of the test itemfor 3min (RT) and 80 min in the incubator (3 inserts per period of incubation time), respectively. 0.996 NaCl (50Nq treated epidermal models were used as negative controls (determination in triplicates) . 8.2.8. Determination of cell viability (MTT) After the incubation period the inserts were washed carefully in PBS (3 times each insert) and MTT reduction was performed. For viability testing the inserts were placed in new 24 well plates containing 300 pi of MTT solution (37'C,1 mg/ml In MTT-assay medium, delivered by Cell Systemso~. The tissues were incubated for about 2 hours under cell culture conditions (5%COz, 37"C, max humidity) . The extraction of blue formazan was performed In Isopropanol (24 well plates, 2 ml / insert) on a vertical shaker (at least 80~ min) . For de(ermination of cell viability the absorption of the Isopropanol-extracts were measured in duplicates at 570 nm in an automatic reader (EL808, Bio-Tek; 98 well format, 200 Nq. Data acquisition and evaluation were done with "KC4" (software by Blo-Tek) . The MTT reduction assay is the most frequently used assay for the determination of cell viability. The assay depends on the intracellular capacity of living cells to chemically reduce the yellow3-[4,5-Dimethythiazol-2-yl]-2,5-diphenyl-tetrazollum bromide (Sigma, Deisenhofen-Germany) to blue formazan crystals. The test has shown to give accurate and reproducible results In various laboratories (2) and has practically been modified for accurate analysis of cell viability in three dimensional skin models. 

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### 6

.2.7. Reliability Chedc , Reliability of the test was confirmed before by interlaboratory Validation (d. Appendix 11 .2) . . 

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### 7. RESULTS 7.1 . Cytotoxic indices (viability decrease measured by MTT) Substances are classified as ,corrosive (R34)", if the cell viability of the EST 1000 is decreased by more than 50%after 3 min of Incubation to the test item, or if the cell viability is less than 1596 after 80 min of exposure to the test item . seen fromthe information in the Table below the test not detected as positive (exceeding the LD50value and the 15 , pectively) by the EST-1000 model after an incubation period of 3 min or 80 min. 7.2. Summary of results Compound Cell viability after 3 min 96 Cell viability after

80 min Classification* 103 .78 102.52 Non-Corrosive l NegatIve control 100.00 100.00 Negative Control

" : Classification was done In accordance with the existing guideline draft and Internationally accepted protocols, i .e. evaluation of LD50values after 3 min and/or less than 1596 viability after a 80 min Incubation period. .

s characterised by no significant impact on cell viability after a e m

!n period. 

### 8 . D AND EVALUATION

ould not be labelled as corrosive to skin.



# . 

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## -is8 . REFERENCES ~ (1) OECD (1998) . Test Guidelines Programme: Final Report on the OECD Workshop on Harmonization of Validation and Acceptance Criteria for Alternative Toxicological Test Methods. Hold on Solna, Sweden, 22 - 24 January 1996 (htto:llwww oecd oroleheltest/backerou_ nd .htm) . (2) OECD , (1998) . Harmonized Integrated Hazard Classification System for Human Health and Environmental Effects of Chemical Substances, as endorsed by the 281' Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, November 1998 (http:llwww

.oecd.orglehs/CIass/HCLB.htm) . (3) Worth, A

.P., Fentem J .H., Balls M ., Botham P.A

., Curren R.D., Earl L.K ., Esdail D .J ., Liebsch M

. (1998) . An Evaluation of the Proposed OECD Testing Strategy for Skin Corrosion. ATLA?&, 709-720 . (4) . Young, J .R

., How, M .J ., Walker, A

.P., Worth, W

.M .H. (1988) . Classification as Corrosive or Irritant to Skin of Preparations Containing Acidic or Alkaline Substances, Without Testing on Animals. Toxic In Vitro, .Z (1) pp 19-28 . (5) Patil, S.M

., Patrick, E., Maibach, H .I . (1998) Animal, Human, and In Vitro Test Methods for Predicting Skin Irritation, In: Francis N . Marzulli and Howard I . Maibach (editors) : Dermatotoxiookogy. Fifth Edition ISBN 1-58032-358-8, Chapter 31, 411-438. (8) Fentem, J .H., Archer, G.E.B., Balls, M ., Botham, PA

.., Curren, R

.D., Earl, L.K

., Edsall, D .J., Holzhutter, H.G. and Liebsch, M . (1998) The ECVAM

intemational validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the Management Team . Toxicology in Vitro 12, pp.483 - 524 . ' (7) EU (2000) Official Journal of The European Communities L136/91 of 8 June 2000, Method B.40 Skin Corrosion. 

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### 10

. ABBREVIATIONS 0 .9°j6 NaCl physiological saline solution RHS reconstructed human skin RHE reconstructed human epidermal model MTT Methylthiazoletetnazolium e . C degree Celsius (Centigrade) RT room temperature cf. confer d day h hour PI microlitre Mg milligram ml millilitre nm nanometre cm 2 oentlmetre2 OD optical density i .e. Id sat (which translates to '"that 191 EST-1000 Epidermal Skin Test 1000 Cat .-No. catalogue number 

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### 11 . APPENDIX 11 .1 . Results Table 1 : Tabular summary of the results 

# PUBLIC COPY Sample Test Item Tm Omsfi StdDsv % viability 1-3 control NaCl 0.9% 60 1 .68 0.08 100 .00 7-9 80 1 .72 0 .07 102

.52 10-12 control NaC1 0.996 3 1 .71 0.07 100.00 16-18 3 i 1 .77 0.05 103 .76 ' 8values Figure 1 : Bar charts of the viability

120.00 100.00 80.00 60.00 40.00 20.00 0.00

## -is11 .2. Ceirtiftation of Validation

~ vWVM CwnbH ~ .r~~ u w CERTIFICATE OF VAUDAT .ION . .

Product Desaipdon: Cat0lop No

. : Size: Lot .No . : P t*wf-1090

ki"814IMS11dilt6tKI4

. Kit for validetlon of'skln corrosfomwith In vitro reconstituted human epkl6rmls ~(:5-1024-V 36 Inserts, 2 te*t Items . . EST-60515-001 CeIISystems elotechnoloyle Vertrleb GmbM ascertains that performing skin corrosion testing

. according to Ce1lSystems Validation Kit Manual has led to equal results at CeIISYstens R&Ddepartment and

wlthirr acceptable variations. InteFlalioratoay reprodudbltlty and 1xllapillty . of.results ~uslnq: .E'SST-~1000 Epidermal .Skin Test Are therelbrt~~ tog , I~ Mine skin corroslon tesllnq accordlnp qo CeliSysteies'pfeto,qble. . .

--- - ----- . Date: O .'h0 " :200t1

soon . 

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7 0IN VITROCHROMOSOME ABERRATION TEST WITH CHINESE HAMSTER VT! CELLS 

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StudY Completion Dafis: December 13, 2008

Page 1 of 46

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GLP Cornpilance Stabnwnt Test Item . Study No. : Except for the following deviations, the study was conducted in compliance with the OECD Principles of Good Laboratory Practice as revised in 1997 (ENV/MCJCHEM(98)17j and with the revised German principles of Good Laboratory Practice according to Annex 1 German Chemicals Act (BundeagesetzblA Part 1, No. 40 issued Ju , acoording to the order of the sponsor no test for dobN the vehkge was performed and no test for homogeneity wa sor stated: "The substance characteristics are not suggestive of a rapid reaction with the vehicle. Therefore, formulations should be prepared freshly prior to treatment . For suspensions no test on homopeneIty of the testsubstanos formulations should be conducted. Suspensions should be mixed appropriately prior to administrallonO . 

### mber 4, 2008CONTENTS



### 1 . Signatures 2. Summary 

### 3 . Introduction 

# PUBLIC COPY 6 7 8 4. Material and Methods g 4.1 . Substances 4 .1 .1 . Test substance 4.1 .2. Positive Controls 10 4.2. Study Design 10 4.2.1 . Origin of Cell Line 10 4 .2 .2. Culturing of V79 Cells 10 4.2 .3 . Composition of S9 Mix 11 4.2.4 . Solvent Selection and Solubility 12 4 .2.5. Determination of CyEofioxdcity 12 4.2 .6 . pHand Osmolapty 13 4.2 .7. Treatment Profiocol 13 4 .2.8. Chromosome Preparations 14 4.3 . Pre-Tents for Cybftxlclty 15 4.4 . Concentrations and Exposure Conditions 18 4 .5. Evaluation Criteria 1 9 4 .8. Biometry 21 4.7. Assessment Criteft 21 4 .8. Assay Acceptance Criteria 22 4.9 . Study Guidelines 22 4.10 . Study Identification and Responsibilities 22 4 .10.1 . Type of Test and Study Number 4.10.2. Responsibilities 22 22 5. Results 5.1 . General Remarks 23 23 5 .2. Mitotic Index 23 5.3 . Survival Index 23 5.4. Chromosome Aberrations 24 

### 8 " Conclusion 254 7. References 26 8. Historical Controls 27 9 . Statement of GLP Compliance 38 10 . Abbreviations used in the Tables 40 11 . Tables 1-10 41p. 40



### Qnalitv Aasnranee Stattment Test Item: Study No . : In the relevant laboratory areas the equipment and standard processes used in connection with toxicological short-tern studies were audited periodically by the Quality Assurance (process- based inspections) . Audit reports have been submitted inwriting to the study director and to the laboratory management. Date of Study Phases Date of report to study dlreetor Inspection inspected and/or management Sept OS, 2006 STUDY PLAN Sept 03, 2006 Nov . 16, 2006 PREPARATION/PROCESSING OF SAMPLES, RAW DATA Nov. 16, 2006 (PROCESS BASED) Dec . 13,2W6 - Dea.13, 2006 FIRST DRAFT Dec. 13, 2006 Dec. 13, 2006 FINAL DRAFT Dec. 13, 2006

'I7ia results ofthe study and the methods used have been correctly reported.

. Date: hm_,A

.. It .1 . Sipnatunsa

Study Director

. \ Date



### Section Head:

i 

## DEC I'% = Date

### L Summary

The dasbogenic potential s evaluated in a chromosome aberration test in vitro. Inidally nsse amstsr VT9 cob were exposed in the absence



### of 89 mix for 4hours to concentrations of 2 .5, 5and 10 Nglml of Itures of all concentrations were harvested 18 hours after the tr+eabnent. In addition, cells treated with 10 pg/ml were harvested 30 hours after the beginning of the treatment Without S9 mix an additional experiment

ontinuous treatment for 1 8 hours, harvest at the same time, a centrations of 2.5, 5 and 10 Nglml . 

### Without and with S9 mix no cybofo)dc effects were observed after 4hours and 18 hours treatment Precipitation in the medium occurred at 5 Nglml and above. None of the cultures treated wit e absence and in the presence of S9 mix showed blologicany reievarn ~rxseased numbers of aberrant metaphases. The positive controls mitomycin C and cyclophosphamide induced dasthgenic effects and demonstrated the sensitivity of the test system and the adivity of the used S9 mix. 

### Based on this teat, considered not to be clastogentc for mammalian cells in vitro

.3 . Introduction The In vitno cytogenetic test is a mutagenicity test systemfor the detection of chromosomal aberrations In cultured mammalian cells (Howard et al ., 1972) . The test is designed to detect structural aberrations in cells at their first post.treatment mitosis. Aberrations of the chromatkl type and of the chromosome type are detectable in this assay. Coil cultures are exposed to the test substance both with and without exogenous metabolic activation. This allows assessing the dastogenic potential of the test substance and of Its metabolites. Following e)"suns of cell cultures to the test substance, the cells are treated with Colcemid to arrest themIn a metaphase-like stage of mitosis (c-metaphase) . Cells are then harvested and chromosome preparations are made. The metaphase chromosomes are stained and analyzed for chromosomal aberrations. cells have been used to assess the dastogenic potential of phase chromosomes prepared from cells at two harvest 

### and 30 hours) and two treatment periods (4 and 18 hours) were evaluated. Aberration frequencies were determined and compared to results of negative con- trols. Study Initiation Date : August 31, 2008 Experimental Starting Date : September 19, 2006 _ Start Date : see Experimental Date : December Starting Date Experiment The study plan, 

### specifled by ,re retained In the archives sample of the sibNlty of the sponsor. 

### e storage of a retention r+eference ItemIs In the respon-

### 4 . Mabrial and Methods 4.1 . Substances 4.1 .1 . Test substance Name of test substance Batch number 

### Content : 95.8 96 (analytical result datedAugust 22, 2006) Approved : until December 9, 2006 Optical appearance' : fine-grained black powder Storage : at roorn tem perature Chemical name CAS-No . The batch used was analytically examined prior to study Initiation and was approved for use for the test period. Astability test in the solvent did not reveal signi8cant degradation of the active ingredient. 

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' don not reflect &emWal oomposidon4.1 .2. Positive Controls

Mitomydn C (Fluka), batch 1149026 10805077 and 1289272 12006009, is a cytostatic agent which inhibits DNA synthesis. Even without metabolic activation, it produces crosslinks In the DNA, which then result in chromosome aberrations. The substance was dissolved and diluted in Hanks' balanced salt solution (Seromed) . From the appropriate dilution, 0.2 ml were added to the positive control cultures immediately after a mediumchange resulting In a final concentration of 0 .1 pglml medium for a treatment period of 4 hours and 0.03 Ng/ml for a treatment period of 18 hours. Cydophosphamide represents a known prortxrtagen and bifunctlonal alkylating agent which induces chromosomal damage after metabolic activation. It was used in the form of Endoxan 100 mg Infection vials of dry substance (Baxter Oncology GmbH) . batch 4N120

. Cydophosphamkie was dissolved and diluted in Hanks' balanced saR solution (Seromed) and 0.2 ml of the appropriate dilution were added per culture immediately after a mediumchange resulting in a final concentration of 2 Nglml medium . 4 .2. Study Design 4 .2 .1 . Origin of Cell Line V7 , Damnstadt . The calls arrived at th November 8,1993. 

### 4 .2.2. Culturing of V79Calls Chinese hamster V79 cells can be kept in culture as established cell lines (Kao and Puck, 1987) . The mean generation time of the used cell line is approximately twelve hours. Prior to the start of the study Chinese hamster V79 cells froma frozen permanent, which was stored In liquid nitrogen, were normally grown In 20 ml mediumand 75 cm2 flasks or under comparable conditions. Incubation of the cells was always performed at 37°C In a COrincubator (596 COz) . Unless reported otherwise, cells were grown In medium

containing 1096 fetal calf serum[FCS = fetal bovine serum(FBS)] .11 n 

### As medium, PAA Ready Mix was used. PAA Ready mix is a commercially available by PAA, Paching, Austria and consists of Eagle's minimal essential medium(MEM, Earls) and the following supplements: PAA Ready Mix (1096 FBS) PAA Ready Mix (296 FBS) 196 L-glutamine 196 L-glutamine 196 MEM-vitamins 196 MEM-vitamins 196 MEM

NEAA 196 MEM NEAA 196 PenIStrep 196 Pen/Strop 1096 FBS (=FCS) 296 FBS (=FCS) The karyotype of the V79 cells (modal number of chromosomes: 22) was confirmed on August 31, 2008 and November 3, 2006 . A routine check for mycoplasma was performed on April 24, 2006 (when the used frozen permanent was prepared) . There was no evidence of mycoplasma oontamination. Therefore no check for myoopiasma contamination was needed ior the actual culture. 

### 4 .2 .3. Composition of S9 Mix S9 mix was used for the simulation of the mammalian metabolism . The S9 fraction was isolated In house fromthe livers of Arodor 1254 Induced male Sprague Dawley rats. The used S9 fraction was derived fromthe preparation dated August 22, 2008 (color-code black, protein content 37.4 mg per ml) . Prior to first use, each batch was checked for its metabolizing capacity by using 2 Irg/ml cyclophosphamide; appropriate dastogenic activity was demonstrated. In addition, each batch was tested in parallel for possible contamination, possible cybotoxic effects and possible dastogenlc effects. Only batches without those effects were used. 

### For use, frozen aliquots of the 89 fractlon were slowly thawed and mixed with a cofactor solution (4:6). The S9 mix contained 4096 S9fraction and was kept In refrigerator and used on the same day .Cofactor solution per 25 ml S9 mix:



### Sodiumphosphate buffer (100 mM ; pH 7.4) 15.0 ml MgC6x 81-1z0 40 " 7 mg 81 .5 mg KCI Glucose-8-phosphate (disodiumsalt) W .0 mg NADP (disodiumsalt) 78.8 mg 4 .2.4. Solvent Selection and Solubility The test subs a suitable solvent~which was selected based on the solubil M the following solvents in the order given. If possible, debniz er isM used as the solvent Test substances that are not sufflcien5y soluble in this solvent are dissolved In DMSO, ethanol or acetone and then added to the medium, if this results in a higher final concentration of the test substance in the medium . 

### Prodpitatlo test at 10 Frg/m 4.2.5. Determination of Cybotoxidty 

### observed in the culture mediumof the. preCytotoxic effects of the test substance were assessed in the pre-test as well as in the main-study. Call survival as well as mftdc index were determined in the presence and absence of 89 mix. At the end of the respective Incubation period cells of all cultures of the respective period were trypsinized, and an appropriate dilution was counted using a hemocytometer (improved Neubauer) to determine call survival .



# F~U~LI s not solubls In any appropriate solvent As redelonized water was selected as vehicle. In this be suspended up to 100 mg/ml (see Chapter 4.4) .

2 solvent w11 be used as terminus fisc:hnk,us, even K dw formulation is a suspension or a emulsionThe mitotic Index was determined for all cultures. The number of mitotic cells among a total of 1000 calls per culture was determined. All calls which were not In Interphasewere defined as mbft . In the main study

. cultures with a total InarbatiQ additionally and exclusively used todetermine the cyboto~ddty 4 .2 .6. pH and Osmolality Concentrations of up to 100 Ii mediumIn the pre-test. not change the pH in the The osmolal r+e-test was not changed by concentrations of up to 100 P 4.2 .7. Treatment Protocol 

### Treatment schedule 4 hours treatment: 0 4 18 30 hours treat- medium first second ment change harvest harvest 

## I I I Treatment schedule 18 hours treatment without 89 mbc: 

## I I 0 18 hours 

### treatment harved The "In vitro Mammalian Chromosome Aberration Test" Is a validated test system . Therefore, there Is no need to oonflrmthe results of the flrst teat by an Identical re. Past- The study protocol of the flrst test covers with 89 mix al possible variations of this method. Ther+efore, there is no possibility to performa second tog which differa relevantly fromthe first trial . Therefore, no Independent repeat was needed for the experimental part performed with S9 mix. 

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,;- 9- 3 

# Lf~ ;The general protocol of the test systemwas similar to published procedures (e.g.

Dean and Danford, 1984) . Chinese hamster V79 cells were passaged on the day prior to treatment . Approximately 1 x 10° cells were seeded in 20 ml of mediumper 75 cm? flasks and Incubated as described In section 4.2 .2. All cultures were set up in duplicate. Immediately before treatment, the medium was removed fromthe cultures. For the treatment, the following fresh solutions were added to each flask:

components volum e In ml 

### - S9 mix + S9 mix medium containing 2%FCS 20 19 S9 mix 0 1 test substance solution 0 .2 1 0

.2 The cola were incubated for the respective period at 37" Cas described above. After 4 hours of treatment, the mediumwas removed; the cells wens washed with PBS. 20 ml of fresh medium

containing 10 96 FCS was added to the flasks and the flasks were placed In a COrlncxrbator for the remaining Incubation time. In cultures boated for 18 hours medium

was not removed. 0 .2 mi Colcemid-solution (40 Nglml water) were added to each flask two hours prior to the end of the incubation period to arrest the cells in a metaphase-like stage of mitosis (c-metaphase) . Positive controls (see section 4 .1 .2) and solvent controls (0.2 ml solvent per culture), and, if indicated, untt~eated solvent) were set up In parallel and handled as desai cultures. Untreated controls and solvent controls are u as nepative control s. 

### 4 .2.8. Chromosome Preparations The mediumwas removed fromeach flask and cogs were removed fromthe bottom of the flask by trypsinizadon and suspended in medium . This mediumwas transferred to a centrifuge tube and spun for approximately 5 minutes at 700 rpm . The supernatant was carefully removed.

'~- g- 71-2 ml of a hypotonic solution (0 .496 KCI ; 37'C) was added to the tube. Within 4 minutes, the volume was brought to 6 ml with additional hypotonk : solution and cells were resuspended. The cells were sedimented in the centrifuge as before and the supernatant was removed. A fewdrops of cold (4°C) fixative [ethanol/acetic acid (3:1)] were added and mixed carefully with the cells. The volume was adjusted to 6 ml with the fixative and mixed again with the cells. The mixture was incubated at room temperature for 20 minutes. Cells were pelleted as before and the supernatant was discarded. Cells were again resuspended In fixative as before and centrifuged. Pelleted cells were reauspended carefully in a small volume of fresh fixative. This suspension was dropped onto dean slides. The slides were allowed to dry for at least 2 hours. Thereafter, they were submerged in pure methanol for 3 minutes and stained for 15-20 minutes In 3% Glemse solution. Slides were rinsed twk;e In water and once in acetone and were then kept In xylene for about 30 minutes. The slides were allowed to dry completely and covered. At least two slides were generated per culture. All solutions used during this preparation were freshly prepared each time. The Giemsa solution was filtered before usage. 

### 4 .3. Pre-Tests for Cybotoxl* The selection of the concentrations used for the main study was based on the results of pro-tests In which cells were exposed with and without S9 mix for 4 hours to various concentrations of the test substance. In addition, cells were exposed without S9 mix for 18 hours to various concentrations of the test substance. Duplicate cultures were tested for every concentration and control . As Indicators of cytotoxic effects, miEotic Indices and numbers of surviving cells (survival Index) were used. The mibtic Index was determined by counting a total of 1000



### sul4mudeffits of the solvent controls were set 10096 and compared to celcultures . 

# UIDs- LiSurviving Cella In the Pre-tests Experimental Group 4 Hours Treatment 

### without metabolic,8cdvation

0 't flc ec .v tla n ff~Do a



### Experimental Group IS Hours Treatrmnt without metabolic ELMVNIW I .Lr'(-I "-,---- . 18



### Concentration Harvest Survival In Time In Index ml Hnum In % 0 0 .1 1 10 20 40 80 80 100 0 0 .1 1 10 20 40 80 80 100 

### Concentration In -jig/ml 24

24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 Harvest Time In Hours 100

.0 119 .3 115 .0 119 .8 115 .5 120.3 112.3 138.5 124.8 100 .0 95 .0 95 .9 101 .2 97.9 99.2 92 .1 98.7 91 .3 

### Survival Index In 96 0 18 100.0 10 18 100.0 20 18 99.4 40 18 103 .4 80 18 97.1 80 18 87.9 100 18 77.0



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### Mitotic Index in the Pre-tests 17

Concentretion Harvest Mitotic In Time in Nudei ml Hours in 96 0 0 .1 1 10 20 40 60 80 100 0 0 .1 1 10 20 40 60 80 100 24

24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 24 100

.0 110 .9 108 .2 106.6 109.8 115.3 110.4 115.3 118.0 100.0 94 .2 93.4 96 .7 102.9 97.5 103.7 102.1 103.7 fConcenftWn Harvest Mitotic in Time In Nudei 

### ml Hours in 96 0 10 20 40 60 80 100 18 100.0

18 105.8 18 120.5 18 113

.5 18 109

.0 18 100

.8 18 109

.0 

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### 18

4 .4. Concentrations and Exposure Conditions Based on the results of the pre-tests and the predpitatbn the medium (see Chapter 4

.2.4), the following concentrations wero use for the main study : test groups S9 mix concentration in treatrrnent harvest culture medium time time as ml in hours in hours -1+ 0 4 18

2.5 4 18 a+ 5 4 18 a+ 10 4 18 

### convas mitomydn C - 0 .1 4 18 hos amide + 2 .0 4 18 -I+ 0 4 30 -/+ 10 4 30 test groups S9 mix concentration In treatment harvest 

### culture medium time time as ml in hours in hours - 0 18 18 

### - 2

.5 18 18 - 5 18 18

10 18 18 Po mitom n C 0.03 18 18 

### For this pu MM g o suspended In deionized water by using sonicadon for m nutes. To avoid heatlno the water of the ultrasonic bath to more than 25'C Ice was added for the last 15 minutes. A black suspension was formed which was well applicable with plpettes. The suspension was mixed prior to administration. 

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### 4 .5 . Evaluation Criteria Coded slides were evaluated using a light microscope at a magnificatlon of about 630 . The mitotic Index was determined by counting 1000 cells per culture. The numbers of mieodc and non-mitotic cob were noted. Duplicate cultures were processed and examined. Coded slides were evaluated using a light microscope at a magnification of about 1000 . Chromosomes of approximately 200 metaphases per concentration, 100 metaphases from each of two parallel cultures, were examined. In most cases at least 100assessable metaphasea were present on one slide prepared froman individual culture. Therefore, to back-up slide which was generated routinely fromevery culture was normally not utilized for the evaluation. However, In cases when fewer than 100 assessable motaphases were found on the first slide of a culture, the be*- up slides were evaluated as well until a total of 100 metaphases was reached. Only metaphases containing the modal chromosome number (22) wens analyzed unless exchanges were detected. In this case, metaphases were evaluated even if the chromosome number was not equal to 22 . The classes of structural chromosome damage were defined and recorded by using essentially the terminology of Rlsger and Michaelis (1987) . Both chnxnatid and chromosome-type aberrations were assessed. Chromatid-type absrrstlons are dastogenic effects restricted to one of the two oorresponding chromatids. Chromosometype aberrations are defined as changes expressed In both corresponding sister chromatids at the same locus. The disdncdon between chromatid and chromosome type aberrations was not made for exchanges. 

### The different classes of aberrations are characterized as follows: 1 . Gap: A gap Is an achromatic lesion within a chromatid armwithout obvious dislocation of the chromatid end(s) and smaller than the width of one chromadd. Gaps are fiound on one chromatid ("gap") or on both chromadds at apparently klerntlcal sites ("iso. gap") . The biological relevance of gaps of both types is unclear.

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a- S" 92. Break: A break is deflned as a discontinuity of one chromatid ("break") or both chromadds, at apparently the same locus ("iaobreak"), with dislocation of the chromatid ends. The dislocated chromatid end(s) has (have) to be present within the respective metaphase. In addition, an achromatic lesion within a chromatid arm without obvious dislocation of the chromatid end(s) but larger than the width of one chromatid is also defined as break or as Isobrn3ak, if this occurs In parallel on both chromatids of a chromosome. 

### 3 . Fragment : Fragments are parts of chromosomes without centnomer. A fragment is the result of a break. The corresponding defective chromosome Is not detedable among the chromosomes of the same metaphase. Fragnmuft can be derived from one chromatid ("fragment") or from both corresponding chromatid regions of a chromosome ("isofrsgmenr') . . 4. Deletion: 

### A deletion occurs as the result of a break . In case of a deletion, one chromatid ("deletion") or both corresponding terminal chromatid parts of a chromosome ("isodeletlon") ans .missingwithin the metaphase under assessment . 5 . Exchange: This is an exchange of chromatid-parts between different chromosomes (interchange) or within the same chromosome (intnachange) . 8 . Multiple aberration: Acall was assessed as to contain "multiple aberrations" when five or more structural changes (excluding gaps) occur within one metaphase. In addition to these aberrations, metaphases; showing chromosome disintegration as an Indication of a cyeotoxk: effect were also recorded ff they were observed. They were counted separately and were not induded among the cells that were assessed for aberrations. "Chromosome disintegration" was recorded if fewer than half of the chromosomes reveal characteristic structural features within a given metaphase. Polyploki metaphases observed In a culture In addition to those 100 metaphases analyzed for chromosome aberrations were recorded. The results are shown In Tables 8 and 10 (WW

number of cob = poiypkoid metaphases + 100) . The number of polyploki metaphases was not used for assessment.



## ,~-7D

### 4 .8 . 8iometry analysis was performed by pair-wise com parison and positive control groups to the r~espective ., .

_ . - r . i i The mitotic Index was statistically analyzed (provided that it was reduced compared to the mean of the corresponding solvent control) using the one-sided chhest. The numbers of metaphases with aberrations excluding gaps were compared (provided that these data super+ceded the respective solvent control) . The statistical analysis followed the recommendations outlined by Rkhardson et al . (1989) . The one-sided che-test was used for the statistical evaluation. A difference was considered to be signMicant, if the probability of error was below 5% . 4.7. Assessment Criteria 

### An Increased incidence of gape of both types without concomitant Increase of other aberration types was not considered as indication of a dastogenic effect. A test was considered positive, if there was a relevant and statistically significant Increase In the abenstlon rate. Atest was considered negative, if there was no such Increase at anytime Interval . Atest was also considered negative, Kthere were statistical significant values, which were, however, within the range of historical negative controls. A test was considered equivocal, if there was an Increase above the range of historical negative controls which was statistically significant but not considered relevant, or If an increase occurred, which was considered relevant, but which was not statistically significant4 .8. AssayAcceptance Criteria 

### An assay was acceptable, if there was a biologically relevant Increase In chromosome aberrations Induced by the positive controls and if the numbers of aberrations for the negative controls were in the expected range based on results fromour laboratory and frompublished studies. 4 .9 . Study Guidelines The studywas perfomned aoooniing to at least the following guidelines: Commission Directive 2000V321EC of 19 May 2000 Official Journal ofthe European Communities of 8 .8 .2000, L 138135- L138/42 8.10. Mutagenlcity - In vitro Mammalian Chromosome Aberration Test 

### OECDGuidelines forTesting of Chemicals "In vitro Mammalian Chromosome Aberration Test" Adopted: July 21, 1997, No . 473 United States Environmental Protection Agency Prevention, Pesticides, and Toxic Substances (7101) EPA712-04W223, August 1998 

### Health Effects Test Guidelines OPPTS 870

.5375 - In vitro Mammalian ChromosomeAberration Test 4.10 . Study Identification and Responsibilities 4 .10 .1 . Type of Test and Number Cytogene8cs i

~n 4.10.2 . Responsibilities 

### Head of Toxicology \ Section Head Genetic Toxivo 11ogy Study Director Senior Technicians Head ofArchives Quality Assurance Analystss. iteauits 5.1 . General Remarks 

### In the absence and posed for 4hours mix Chinese hamster V79 cogs were ex10 Nglml and, in , for 18 hours, centrations of up to and Including 10 Nglmi . Without a uding dpitation occurred In the medium at the concentration 51rg/ml and above. 5 .2. Mitotic Index 

### oonpreThe mitotic Indices determined In the main study are listed in Tables 1 (4 hours treatment) and T (18 hours treatment) . Mitotic Index without S9 mix: 

### In comparison to the solvent control, the mitotic Indices in On treated cultures were not relevantly reduced. The cultures treated with mitomydn Cshowed also no reduotion In mibosis rate. Mibotla Indexwith S9 mix: In comparison to the solvent control, the treated cultures showed no relevant neduotlon of the mitosis rate. The positive control cydophosphamide reduced the mitosis rate. 5.3. Survival Index 

### The survival Indices determined In the main study are listed In TablAs 2 (4 hours treatment) and 8 (18 hours treatment) . Survival Index without S9 mix - In comparison to the solvent control, the survival indices in the treated cultures were not nolevantly reduced. The cultures treated with m1tomycin C showed a reduction in survival rate only after 4 hours treatment. 

### Survival indexwith S9 mbc: In comparison to the solvent control, the treated cultures showed no relevant reduction of the survival rate. The positive control cyclophosphamide reduced the survival rate. 

# Ll X73

### 5

.4 . ChromosomeAberrations The results of the micrvsoopic evaluation of the metaphases for structural chromosome aberrations are presented In Tables 3-5 (4 hours treatment) and 9 (18 hours treatment) . Numbers of abenations (listed by don) and numbers of metaphases with aberrations Induding -and excluding gaps for individual cultures are listed (in per - 

### out S9 mbc

: n~levant increases of numbers of metaphases with aberrations were detected alter 4 hours treatment and total culture times of 18 or 30 hours (Tables 3 and 5) . The same was true for a beatrnent period and total culture time of 18 hours (Table 9) . In agreement with our assessment criteria (Chapter 4.7) the statistical significance observed at 10 Ng/ml after 4 hours treatment and total culture times of 18 hours is considered to be of no biological relevance since it was within the range of the attached historical controls (see Chapter 8) . 

### The treatrnent with the positive control mitomy~cin C resulted In a dear and statlsdcally significant increase of metaphasss with aberrations and demonstrated the sonOf thetest system

. S9 mbc: 

### nt and statistically significant increases of numbers of metaphases with aben~ations were detected ~4 hours treatment and total culture times of 18 or 30 hours (Tables 4 and 5) . The positive control cydophosphamide Induced statistically signific~nt and biologIcally relevant Increases of metaphasss with aberrations and demonstrated the sensitivity of the test system

and the activity of the used 89 mix. 

### cent)-

### 6

. Conclusion ARer 4hours treatment of Chinese hamster V79 cells ©erbations of 2

.5, 5 and 10 N

dm! wa u nd of the 

### ment 

### ment addition, after .1 S hours treat- .5, 5and 10 Irglml were selected for reading None of these cultures treated e absence or presence of S9 mix showed biologically relevant inc ers of metaphases with aberrations. 

### The positive controls mi6omycin Cand cydophosphamide induced dear dastogenic effects and demonstrated the sons" of the test systemand in the case of cydophosphamide the a*vity of the used S9 mix. Based on the results of this JIMMOonsidered not to be dastogenic for mammalian cells In Vitro. 

# PUBLIC COPiv

### T. References 1 . Ames, B.N., Dun;ton, W .E., Yamasaki, E. and Lee, F.D.(1973) Carcinogens are mutagens: Asimple test system combining Ilver homogenates for activation and bacteria fbr detection Proc. Not . Aced. Sd. U.S.A . 70,2281-2285 2. Ames, B.N., AAcCann, J . and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonellalmarnmalian-micr+roeome mutagenicity test Mutation Res. 31, 347-384 3. Dean, B.J . and Danfad, N . (1984) Assays for the detection of chemically-Induced chromosome damage In cultured mammalian cells 

### in: Mutagenlcity tesdng - a practical approach, (ad . Venitt, S . and Parry, J . M

.) . IRL Press, Oxford 4 . Hawani, P

., Bloom, A

.D. and Knooth, R

.S. (1972) In vitro 7, 359-385 5 . Kao, F.T. and Puck, T.T. (1987) Genetics of somatic mammalian cob. IV . Properties of Chinese hamster cell mutants with respect to the requirement for prollne Genetics 55, 513-518 8 . Richardson, C

., Williams, DA

., Man. JA

., Amphlett, G

., Chanter, D

.O . and Phillips, B. (1989) 

### Analysis of data fromIn vitro cytogeneticc assays in: Statistical evaluation of mutagenkty teat data, (ad. D .J . Kirkland), Cambridge University Press, Cambridge 7. Rieger, R. and Michaeiis, A . (1967) Die Chromosomenmutationen VEB Gustav Fischer Verlag, Jena 

# PUBLIC u'o--27



### e. Hlstodcal Controls 4 Hous Treahtak Cytogarndcs M

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# PUBLIC Cp. 64

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# PUBLI

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### 9 : gap 19 : iso9aP b : break 

### ib : isobroak f : fmgment lf : isofragment d : deietbn Id : isodeWon ex :exchange ma : multiple abomdIons 

### maE : mulWe abeRadonswith exchange cd : chromosome disintegmtlonp. 76

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### SALMONELLAIMICROSOME TEST PLATE INCORPORATIONAND PREINCUBATION METHOD

Study Completion Date: December 15, 2008

Page 1 of 56 

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### OLPCompliance Statement Test Item Study No . \

Except for the following deviations the study was conducted in compliance with the OECD Principles of Good Laboratory Practice as revised In 1997 [ENVIMC/CHEM(98)17] and with the revised German principles of Good Laboratory Practice according to Annex 1 German Chemicals Act (Bundesgesetzblatt, Part I, No. 40 issued June 27, 2002) . As requested by the sponsor, no analysis was per- formed on stability of the test itemin the vehicle and no test was performed concern_ Ing the homogeneity of the test substance In formuladon. However, the sponsor stated: "The test substance characteristics are not suggestive of a rapid reaction with the vehicle. Therefore, formulations should be prepared freshly prior to-start of treat- ment" .

ecember 10, 2008 

# PUBLIC COPYCONTENTS



### 1 . Signature 8 2. Summary 7 

### 3 . Introduction 8 4. Material and Methods 10 4 :1 . Substances 10 4 .1 .1 . Test substance 10 4 .1 .2. Positive Controls 11 4.2 . Indicator Organisms 11 4.2 .1 . Description of Test Strains 11 4.2.2 . Origin of Strains 13 4.2.3 . Production of Stock Cultures 13 4.2.4. Checking the Genotype of Stock Cultures 14 4.2.4.1 . Hisddine Requirement . 14 4.2 .4.2 . Ampidllin Resistance (pKM101) 14 4.2 .4.3. Tetracycline Resistance (pAQ1) 14 4.2.4.4. Crystal-Violet Sensitivity (deep rough) Is 4 .2.4.5. UV Sensitivity (uvrB) 15 4 .2.5. Stock Batches 15 4 .3. S9 Mix ' 16 4 .4. Test Design . 17 4 .5. Assessment Criteria 21 4 .8 . Study Guidelines 22 4.7. Study IdentIfIcatIon and Responsibilities 22 4.7.1 . Type of Test and Study Number . 22 4.7.2 . Responsibilities , 22 

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### on a arosome Test 4

5. Results 23 5.1 . Description of Results . 23 5 .2 . Tabulated Summary of Data 24 8 . Assessment 26 7 . References 27 8. Historical Controls 30 8.1 . Plate Incorporation Method 30 8.2. Preincxrbadon Method 38 9 . Statement of GLP Compliance 46 10 . Abbreviations used In the Tables 48 11 . Tables 1-10 ' 49 

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### 4u"h Assurmnce S_tstement Test Item: Stud,y~No . : In the relevant laboratory areas the equipment and standard processes used in connection . with toxicological short-term studies were audited periodically by the Quality Assurance (process- based inspections) . Audit reports have been submitted in writing to the study director and to the laboratory managenent . Date of Study phases Date of report to study director Inspection inspected and/or managenent Jul . 17, 2006 STUDY PLAN Jul . 17, 2006 Nov. 30, 2006 PREPARATIONOF FORMULATIONJFEEDMIXTURE, RAW Nov . 30, 2006 DATA (PROCESS BASED) Dec. 14, 2006 - . Dec . 14, 2006 FIRST DRAFT Dec . 14, 2006 Dec . 14, 2006 FINAL DRAFT Dec . 14, 2006 

# PUBLIC COPY

The rpmdWfthe study and the methods used have been correctly reported. Date: Ice_ Aft, Aft`



## 3~- ~1 . Signature

Study Director

. December 15, 2008 

### Date 

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### Z. Summary 

### a s initially Investigated using the Salmoneila/microsome plate ncorpora~on test for point mutagenic effects in doses of up to and Including 5000 Nn per plate on five Saimoneiia typhimurtumLT2 mutants. These comprised the his- tidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102 . The inde- pendent repeat was performed as preinarbation for 20 minutes at 37°C . Other condi- tions remained unchanged. Doses up to and including 5000 Ng per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation occurred at the dose 158 irg per plate and above. Evidence of mutagenic activity as not seen. No biologically relevant increase in the mutant coun , n comparison with the negative controls, was observed. The positive controls sodium azide, nitrofurantoin, 4nitro-1,2-phenylene diamihe, mitomydn C, cumene hydroperoxide and 2-aminoanthracene had a marked muta- genic effect, as was seen by a biologically relevant Increase In mutant colonies com- ---- , . _ . . din 

### Therefo S9 mix In Salmonella/mlcrosome test . 

### considered to be non-mutagenic without and with as well as In the preincubation modiflcation of the 

# PUBLIC COPYmonellalMicrosome Test 

### 8

3 . Introducdon The evaluation of the mutagenicity of the test substance was pe6rmed using the Salmonella/microsome test, also termed the Ames test, as described by Ames et al . (1973a, 1975) and Maron and Ames (1983) . The Salmonella/microsome test is a screening method which detects point mutation caused by chemical agents in vitro . Auxotrophic mutants of Salmonella typhimurium are used to demonstrate this effect. For this purpose, the rate of reversion to proto- trophy Is evaluated In negative control and treated groups. A mutagenic effect Is as- sumed if this rate Increases sufficiently in the treated groups. Mammalian metabolism, which is of great significance In chemical mutagenesis, is simulated in this test by the 9000 g fraction of homogenized mammalian livers. To- gether with co-factors, this forms the "S9 mix" which represents the metabolic model in this test. The method Itself Is considered to be very sensitive (Herbold et al ., 1976; Herbold, 1978) and is well suited for fast screening. Available literature indicates a high corre- lation between the positive and negative responses of the Ames assay and the car- cinogenic activity of the tested substances (McCann et al ., 1975a, 1978 ; Purchase et alr, 1976, 1978) . In addition, the test represents a good screening system for poten- gal carcinogenic effects, although the results should not be overrated, as this high correlation may not apply to all substance groups (Ames, 1979 ; Andrews et al ., 1978

; Clayson, 1980

; Glatt et al ., 1979 and Rinkus and Legator,1979; Zeiger, 1987) . 

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# 491wmee,M °

Study Initiation Date : July 6, 2008 Experttnental Starting Date : November 28, 2006 Study Start Date : see Experimental Starting Date Experimental Completion Date : December 7, 2005 fi th 

### report are retained In the archives specified by item and, if applicd sor. e storage of a retention sample of the test of the reference itemis in the responsibility of the spon

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### 4 . Material and Methods 4 .1 . Substances 4.1 .1 . Test substance Name of test substance Batch number 

### Content 0 .11A Hydrogen 0.5 96 Nitrogen 0.7 96 Cobalt 0.5 96 Aluminum 0.6 96Magnesium 0.5 96 Manganese (analytical result dated August 22, 2006) 

### Approved until December 9, 2008 Optical appearance' : black powder 

### Storage : at room temperature Chemical name Indication : composite component The batch used was analytically examined prior to study Initiation and was approved for use for the test period. 

# PUBLIC COPY 11 does not refled chemicel composition4 .1 .2. Positive Controls 

### Sodiumazide (Na-azide, SERVA), order no. 30175, a direct-acting mutagen used as specific positive control for TA 1535. Nitnofurantoin (NF, SIGMA) ; order no . N-7878, lot no

. 014K1284, a directacting mutagen used as specific positive control for TA 100 . 4=Nitro-1,2-phenylene diamine (4-NPDA; Merck-Schuchardt), order no . 806294, batch no. S20772711, a direct-acting mutagen used as speciflc positive control for TA 1537 and TA 98. 

### Mitomycin C (MMC, Fluka), order no. 69824, lot &fllling number 1289272112006009, a directacting mutagen used as speciflc positive control for TA 102 in plate Incorporatlon trials. Cumene hydroperoxide (Cumene, Sigma), order no . C-0524, lot no. 78H0879, a direct-acting mutagen used as specific positive control for TA 102 in preincubation trials. 2-Amlnoanthracene (2-AA, Aldrich), order no. A3,880-0, batch no . 11997-070, a promutagen which reverts all the strains and serves as a control for the activating effect of the S9 mix. ' The positive controls sodium azide, nitnftrantoin, 4-nitro-1,2-phenylene dlamine, mitomydn C and cumene hydroperoxide were only used without S9 mix; the positive control 2-aminoanthracene was only used with S9 mix. 

### 4 .2. Indicator Organisms 4.2 .1 . Description of Test Strains Histidine-defldent mutants of Salmonella typhimurlum LT2 served as Indicators to demonstrate point mutagenic effects. These strains were selected specifically for the Salmonella/ microsome test. Since point mutations can be divided into two basic classes, base-pair substitutions and frameshiR mutations, several strains were used which cover both types. 

# PUBLIC COPYmonellalMicrosome Test 

### 12

Thesq included the strains selected by Ames et al . (1973b), Salmonella typhimurium TA 1535 and TA 1537, as ~well as Salmonella typhimurium TA 100, TA 98 and TA 102 developed by McCann et al . (1975b) and Levin et al . (1982), respectively. TA 1535 and TA 100 bear the base-pair substitution, his G46, and TA 100 additionally contains the plasmid pKM101 . This R factor also contained in TA 98 and TA 102, codes for an ampicillin resistance and should raise the sensitivity of the strains. TA 102 carries the ochre mutation his G 428 on the multicopy plasmid pAQ1, which codes in addition for tetracycline resistance. TA 1537 and TA 98 bear frameshift markers. TA 1537 exhibits the +1 mutant, his C 3076, while TA 98 bears the +2 type, his D 3052

. . Furthermore, the strains have other properties, which should increase their sensitivity. They are all deep rough, i .e. partly deficient In lipopolysaccharide side chains in their cell walls, enabling larger molecules to penetrate the bacterial cell wall and produce mutations. With the exception of TA 102, all strains have reduced capability to repair DNA-damage which Increases the likelihood that such damage results in mutations. ' Whereas TA 1535 was used in addition to TA 100, TA 1538 is not normally used In addition to TA 98

. This has two reasons: 1 . There Is no relevant increase in the spontaneous mutant counts of TA 98, compared to the spontaneous range of TA 1538. Special difterences in sensitivity existing between TA 1535 and TA 100, arid attributed to the relatively high spontaneous rate of TA 100 (10 times that of TA 1535), do not exist between TA 1538 and TA 98. 2. An International general inquiry had shown that using TA 1538 in addition to any of the test strains in this study would not provide further information of biological relevance (Herbold, 1983) . This Is In agreement with International guidelines, as published by OECD, EEC, or EPA . Strain TA 1538 was either deleted in these guidelines, or never Introduced at all . Maron and Ames (1983) also reported: "Although TA 1538 is useful for the detecdon of particular aromatic frameshift mutagens such as 4nitro-o-phenylene diamine, we decided to drop the strain because it overlaps considerably with TA 98" . 

# PUBLIC COPY -3)- faImoneIla/Microsome Test 13

TA 1538, which differs fromTA 98 in lacking the piasmid pKM 101, Is used in spite of these considerations, if questionable TA 98-results need clarification. This was not the case in the present Investigation, however. 

### 4 .2.2 . Origin of Strains original strains were obtained from P rof. Bruce Ames and arrived 4 .2.3. Production of Stock Cultures Immediately upon receipt the samples were inoculated on nutrient agar plates, to which ampicillin had been added for the TA 100, TA 98 and TA 102 strains. The plates for TA 102 contained additionally tetracycline. These plates were Incubated at 37°Cfor approximately 24 hours. Samples were taken fromindividual colonies with a sterile Inoculation loop, and transferred to nutrient broth. In the case of TA 100, TA 98 and TA 102 ampicillin had also been added to this broth. The broth for TA 102 contained additionally tetracycline. The samples were again incubated overnight at 37°C . Newsamples of these cultures were Inoculated onto nutrient agar plates, which had again been provided with ampicillin for TA 100, TA 98 and TA 102 and In addition with tetracycline for TA 102. After an incubation period of approximately 24 hours at 37°C, new samples of indi- vidual colonies fromthese plates were transferred to flasks containing approximately 20 ml normal nutrient broth. This inocuium was incubated overnight at 37°C, after which a small sample was taken to check the genotype. At the same time, the re- maining cultures were treated with DMSOto protect against the effects of freezing, and Immediately frozen at -80°C in ~1 ml portions (Ames et al ., 1973b; McCann et al ., 1975b) . No ampidllin- or tetracycline-resistance test was done on the samples used for testing genotype since the cultures had already been sufficiently selected by am- picillin and tetracycline. In addition to the test for crystal-violet sensitivity (deep rough character), a test was done for UVsensitivity (uvrB) where approprIate. .The crystal-violet and UVsensitivity tests are described below . The frozen cultures which did not produce satisfactory results here were discarded. Remaining cultures were stored for future tests. In addi- tfon, frozen cultures of batches with unsatisfactory negative and/or positive control results in the definitive tests were also discarded.



# PUBLIC COPY 32-7Whenever newstock cultures needed to be produced, individual cultures grown on nutrient agar were used, to which ampicillin had been added for the TA 100, TA 98 strains and to which ampicillin and tetracycline was added for strain TA 102. Samples of these Individual colonies were then transferred to approximately 20 ml nutrient broth, Incubated, divided up, and checked for crystal-violet and UN sensitivity, if appropriate. One 1 ml-por8on was thawed for each test and strain, and quantities of 0.2 ml of the thawed culture were added to 10 ml nutrient broth. This culture was Incubated overnight at 37°C and used only on the same day. A new, small stock culture, which had been checked for its properties directly before freezing, was thus available for each Individual test . In general this obviated any need to re-check the genotype for each Salmbnella/mlcxosome test. This procedure is In accordance with the methods described by Ames et al . (1975) and Maron and Ames (1983) . 4.2 .4 . Checking the Genotype of Stock Cultures 4 .2.4.1 . Histldlne Requirement A special test fo'r histidine requirement was not necessary since histidine dependence of the cultures was automatically checked by the accompanying negative controls In each indlvlduai test of this study. The number of mutants per Individual plate Is listed In the Tables ~1 to 10 . 4.2.4.2. Amplcillln Resistance (pKM101) 

### Aspedal test for ampicillin resistance was not necessary since strains TA 100, TA 98 and TA 102 were Incubated on ampicillin containing nutrient agar and formed Individual colonies. Consequently surviving bacteria were ampicillin resistant . 4.2.4 .3. Tetracycline Resistance (pAQ1) A special test for tetracycline resistance was not necessary since TA 102 was Incubated on nutrient agar containing In addition to ampicillin tetracycline and formed Individual colonies. Consequently surviving bacteria were also tetracycline resistant.



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### a mone e Test 15

4.2.4 .4. Crystal-Violet Sensitivity (deep rough) A quantity of 0

.1 ml was taken fromthe samples of individual stocks and spread onto nutrient agar, using four plates per strain. After a few minutes, 51ter papers, to which 10Irl of an aqueous, crystal-violet soludon had been added at a conoentretion of 1 mg/ml, were placed in the middle of the plates. The plates were then incubated overnight at 37°C

. The diameters of the inhibition zones that had formed were then measured. The inhibition zones of all stock batches used Indicated an adequate sensitivity to crystal-violet. 4 .2.4.5 . UV Sensitivity (uvr8) As described under 4.2

.4 .4, samples were spread onto nutrient agar plates. One half of each plate was covered with aluminumfoil and irradiated without a lid for six seconds (TA 1535 and TA 1537) or eight seconds (TA 100 and TA 98) with UV light of a wavelength of 254 nmat a distance of 33 cm . The Irradiated plates were incubated as described under 4.2.4.4 and checked. To demonstrate adequate sons" in this test, cultures had to show an Inhibition of growth over haff their area, i .e. no bacteria should have grown on the Irradiated half. This was the case with all the stock batches used. For TA 102 no such test Is needed. 4 .2 .5. Stock Batcheff

Stock Batches Used In Tables Strain 1-5 6-10 03.05 .0613 03

.05.06/3 . TA 1535 03.05.06/2 03.05 .0611 TA 100 03 .05.06/3 03.05 .06/3 TA 1537 03 .05 .06/1 . 03.05.06/4 TA 98 03.05 .06/2 03

.05.06/1 TA 102 

# PUBLIC COPY4.3 . S9 Mix S9 mix was used to simulate the mammalian metabolism of the test substance. It was made fromthe livers of at least six adult male Sprague Dawley rats, of approximately 200 to 300 g In weight. For enzyme induction, the animals received a single intraperltoneal Injection of Aroclor 1254, dissolved in corn oil, at a dose of 500 mg/kg body weight, five days prior to sacrifi©e. The animals were prepared unfasted, following the directions of Ames et al . (1975) and Maron and Ames (1983) . The rats. were terminated. Livers were removed under sterile conditions immediately after sacrifice and kept at 4°C until all animals had been prepared. All the remaining steps were carried out under sterile conditions at 4°C . . The livers were washed with cold (4°C), 0 .15 M KCI soludoh (approximately 1 ml KCI per 1 g liver), . and then homogenized in fresh, cold (4°C), 0 .15 M KCI (approximately 3 ml KCI per 1 g liver) . The homogenate was then centrifuged in a cooling centrifuge at 4'C and 9000 g for 10 minutes. The supernatant (the S9fraction) was stored at -80°C in small portions. , 

### These portions were slowly thawed before use . The S9 mix was freshly prepared (Ames et al ., 1973a), kept on Iceand used only on the same day. Seventy ml of cofactor solution are composed as follows: MgC6x 8 H20 182.6 mg KCI 246 .0 mg Glucose-8-phosphate, disodiumsalt 179 .1 mg NADP, disodiumsalt 315.0 mg Phosphate buffer 100.0 mm S9 mix consists of this cofactor solution, S9fracdon and, If needed, 0 .15 M KCI . The amount of S9fraction In S9 mix is Indicated In the tables In percent. The SOmix comprised the amount of S9fraction (x°16) Indicated in the tables, 70%cofactor soiutlon and (30-x)°X, 0

.15 M KCI . The S9fraction was derived fromthe preparation dated 

### August 22, 2000

.(protein content 37.6 tng per ml) . 

# PUBLIC COPY 33)~-'Prior to first use, each batch was chedced for its metabolizing capacity by using reference 'mutagens; appropriate activity was demonstrated. At the beginning of each experiment 4 aliquots of the S9 mix were plated (0.5 ml per plate) in order to assess its sterility. This was repeated after completion of test tube plating. The sterility control plates were then incubated for 48hours at 37°G: No Indication of contamination of S9 mix was found. 4.4. Test Design The initial plate incorporation test foiiowed the directions of Ames et al . (1973a, 1975) and Maron and Ames (1983) . Plate Invorporeflon rest

: 1,W"A~ twh"Aft 9801"Awd?

ftftr A W"N"W bmaftw MON"

"Mao so at 4SV . OW 4W &sub~ -MVM=f&*" MA So am a*~ AWAMW

df &" a AV $8&a For the mutant count, three plates were used, both viilth and without S9 mix, for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control . Each positive control also contained three plates per strain. In experiments without S9 mix buffer was used as replacement . 

### Generally, the amount of solvent for the test substance and for . the controls Is 0

.1 ml In the present study the amount of solvent per plate used for nd for the negative controls was 0 .5 ml per plate due to the low 

### the test substance. 

# PUBLIC

. COPYRtrwau+. 41 JW WAWMN

IMbMwr Rf dWOMrWIww The doses for the first trial were routinely determined on-the basis of a standard protocol : if not limited by solubility 5000jig or 5 PI per plate were used as the highest dose. At least four additional doses were routinely used. If less than three doses were used for assessment, at least two repeats were pertormed. The results of the flrst experiment were then conaidered as a pre-test for toxicity. However, in case of a positive resporise or If 9t least three doses could be used for assessment, the first 

### trial was Included in the assessment . If the second. test confirmed the results of the first, no additional repeat was performed . Doses of repeats were chosen on the basis of the results obtained in thefirst experiment . The independent repeat was performed as preincubaflon In a water bath at 37°Cfor 20 minutes. At the end of the preincubatfon period 2 ml of molten soft agar were added to the tubes, the content mixed and plated. Prelflombetlon root Rr .aRqr

uaftw wrrri

4 .r rwkr r

, ar'elYrwtAnr For the mutant count . three plates were used for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control . Each positive control also contained three plates per straln. The doses of this trial were determined on the basis of the results of the plate Incorporation assay. Doses are given as jig/tube for better separation of plate IncorporaHon and preincubatian trials, despite the fact that jig/plate and Vg/tube could be used synonymously. . "L :'3?za AraArs, .w *WOW ' whr .w . . " An 

### 18

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# PUBLIC COPY19

The toxicity of the substance was assessed in two ways. The first method was, a gross appraisal of background growth on the plates for mutant determination. If, a reduction In background growth was observed, it was Indicated in the tables by the letter "b" after the mutant count. Where only a single "b", without any other values, Is noted for e concentration, this "b" represents three plates with reduced background growth. (The same applies to the signs "o", "v", "p", "n" or "96", which may also be used In the tables) . Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. The bacterial suspensions were obtained* from 17-hour cultures In nutrient broth, which had been incubated at 37°C and 90 rpm . These suspensions were used for the determination of mutant counts. No standardized procedure was employed to set the bacterial suspensions at a defined density of viable cells per milliliter, since the chosen method of incubation normally produces the desired density. However, the num- bers of viable cells were established in a parallel procedure by determining the fiters of the negative controls. The dilution of bacterial suspensions used for the determination of titers was 1 :1,000,000. Titers were determined under the same conditions as were the muta- tions, except that the histldine concentration In the soft agar was Increased five-fold to permit the complete growth of bacteria. Total bacterial counts were taken with S9 mix on two plates for each strain. However, If an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix. The results of these determinations may be seen In the negative controls in Tables 1 to 10 . ~ The tests were performed both with and without S9 mix. Full details are given In the Tables 1 to 10

. The count was made after the plates had been Incubated for 48 hours at 37°C . If no immediate count was possible, plates were temporarily stoned in a refrigerator. If not Interfered e.g. by precipitation on the plates or coloration of the plates, colonies were counted automatically using an Artek counter, model 982B. Data were transferred to a PC and processed with the released and DOS 6.0 based BioSys software Ames- Test III (Rev. 3.1.06) . 

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