, PUBLIC . COPY 

l . Signatures 

Study Director. t v 

Date 

Management.- ,Z, 1144b%, . 20o b 

Date 

J 
, PUBLIC CO 

2. Summary 

This study was ed to assess tential irritant / corrosive effects of 

'ty: 95. n the skin of rabbits. 

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 posidw rosponse : 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 2 days. ' . 

According to~classi8cation crlte not an lrritant to the 

skin.' 

Conclusion: 

There were no systemic intolerance reaction:. 

The method used complied with the OECD - Guideline for Testing of Chemicals 

No. 404 -" Acute Dermal Irritation/Corrosion"; adopted: 24th April 2002; EEC 

Directive 67/548 Annex V - Method H.4 . (1967) in its current version. 
, 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) 

Study completion date : see signature page 

The study objective was to determine the irritant / corrosive effects on akin of albino 

rabbits . Information derived from this test serves to indicate the possible existence 

of hazards likely to arise from short-tam exposurE of akin to the test substance, 

and - with respect to a proper handling and use - serves to permit classification . 

~(labelling) of a product. 

The st~~w~ n1an- raw data and the final report are retained in the archives specified by 

e storage of a 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 of Test Facility 

' Head of Department 

Archiving 

Head of Quality Assurance Unit 

PU 
40 , PU 

5. Material and Methods 

LIC COPY 

The method used complied with the OECD - Guideline for Testing of Chemicals 

No. 404 -" Acute Dermal Irritation/Corrosion"; adopted: 24th April 2002; EEC 

Directive 67/548 Annex V - Method H.4. (1967) in its current version. 

For reasons of animal welfare a sequential testing strategy was followed in -

accordance with the current version of the EEC Directive 67/548 and the OECD 

Guideline No. 404, irrespective of the requirements of other 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 performance of a SAR evaluation for sldn corrosion/irritation, 

measurement of pH value, the evaluation of data on systemic toxicity via the dermal 

route and the performance of a validated in vitro test for skin corrosion (Human 3D' 

Epidermal Skin Model) before in vivo testing for skin irritation/corrosion in rabbits. 

The in vitro test for skin corrosion is archived J"~be test compound 

is not corrosive to the skin. 
 PUBLIC CO 

5.1 Test Compound 

Test compound 

Chemical name 

Appearance : black, powdery to coarse-grained 

Storage : room temperature 

Batch no. 

600900b 

Content of test substance : 95.8 t used for calculations) 

Expiry date : 2006-12-09 (YYYY-MM-DD) 

Confirmation of the identity of the test compound was performed: ' 

5.2 Test System (Anlmals) 

The study was performed in female albino rabbits (strain Crl:KBL(NZW)BR, 

Charles River, 88353 KiBlegg, Gummy) recommended as the preferred species for 

this type of studies. 

Healthy adult albino rabbits fme of clinical signs were used. The health of the 

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 of the 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 of this study. 
11 PUBLIC COPY 

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 

The animals 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, 73494 Rosenberg, Germany). The wood granulate was 

changed at. least twice weekly. The animals were regularly transferred to clean 

cages. 

The animal room had a standardized climate: 

Room temperature ' : 20 }3°C 

Air humidity : 50 :k25% 

Light/ Dark cycle : 12 hours rhythm. 

The humidity and air temperature were continuously recorded. Occasionally 

deviations from the standards occurred, e.g. during cleaning of the animal room or 

effects of the weather. They did not have any apparent influence on the outcome of 

the study. ' . 
 PUBLIC - COP 

The animals received the standard diet "Ssniff K-Z" 4mm (manufacturer: Ssniff 

Spezialdifiten GmbH, 59494 Soest, Germany), approximately 100 g per animal per 

day ~and tap water ad libitum from polycarbonate bottles. 

The nutritive composition and the contaminant content of the standard diet were 

checked and analyzed routinely in random samples. Nothing untoward was found. 

The tap water was of drinking water quality (according to the Drinking Water 

. Decree in the current version) and analyzed routinely. The wood granulate was 

randomly checked for contaminants at regular intervals. 

The results of these analyses have been ato 

_ e available data yielded no evidence of any adverse 

. effects on the aim of the study. . 

The animal room was cleaned at least once a week and disinfected at least once a 

month. It was ensured that the diet was not contaminated, and that there was no 

contact of the 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 from the 

dorso-lateral area of the trunk of each of the rabbits. Care was taken to avoid 

abrading the skin. Only animals with healthy and intact skin were used. 

0.5, g of the pulverized test substance moistened with water (to ensure good contact 

with the skin) was applied to the skin of the animal under a gauze patch. The treated 

skin area was approximately 2.5 cm by 2.5 cm in size. The patch was placed on the 

dorso-lateral areas of the trunk of each animal and was held in place with nonirritating tape for the duration of the exposure period. Thus, access by the animal to 

the patch and resultant ingestion of the test substance was prevented. 
13 PUBLIC C 

'Mar the exposure period the dressing and patch were removed. The exposed skin 

area was carefully washed with water without altering the existing response, or the 

integrity of the epidermis. The surrounding untreated sldn served as control. 

Due to a possible irritant potential of the 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 patch was removed after three minutes. As no serious 

skin reactions were observed, the second patch was applied and removed after one 

hour. At this stage the observations 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, animals were 

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. 

The degree of erythema/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 and fully described., 

As general criteria the body weight of each animal was recorded at the beginning of 

the study. If clinical findings other than dermal irritations occur they were recorded 

daily. 
p. 10 

14 

PU LIC 

5.6 Evaluation and Interpretation of Results 

The interpretation of the results is based on the directive 67/548/EEC and its 

subsequent amendments (see Appendix Table 8-2). 
6. Results 

The individual findings of the treated s1dn areas at the various observation times are 

summarized in the tables below. The control 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 from the akin, slightly block discoloration 

Table 6-2 Irritant Effects on the Skin (Exposure: 4 hours) 

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") an a aONr 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, 1 h": test compound could not be removed completely from the skin, 

1 h - 24h': slightly black discoloration of the treated skin 

Animal 2, 1 h': test compound could not be removed completely from the skin, 

, 1 h": slightly black discoloration of the treated skin 

Animal 3, 1 h": test compound could not be removed completely from the skin, 

1 h - 24h": slightly black discoloration of the treated skin 

' s after patch removal 

}-y 6 
p. 12 

16 

PUBLIC C 

Table 6-3 Summary of Irritant Effects (Score) 

. Mean Reversible 

Animal 24 h 48 h 72 h scores Response (days) 

EMhema (rodness) 

1 and esdvar formation 0 . 0 0 0.0 - no 

pederne fonrsMon __ 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 2 days. 
7. Conclusion - 

The irritant / corrosive potential o 

412MIwas 

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; EEC Directive .67/S48 Annex V - Method B.4 . (1'967) in its current version 

According to classification crite 

skin. , 

ot an irritant to the 

There were 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 eschar formation preventing grading of 4 

erythema 

Maximum possible: 4 

Oedema Formation 

, No oedema - 0 

Very slight oedema (barely perceptible) 1 

Slipht oedema (edges of area 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) 

Maximum possible: 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 . UBLIC C 

interpretation of Results 

Table 8-2 Interpretation of Results 

Causes severe burns 

A test 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 to four hours exposure. ~ 

IrrItating to skin 

A test material with Irritating potential to skin is a test material that produces 

at least in 2 of 3 tested 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 

20 

PUBLIC COPY 

ft Umwelt und Nat~u~sch Landwirtschaft und 

Verbraucherschutz 

des Lande,s Notdrhein-W en 

Nft+ekrJC 10190 AOwkloef Aiawm~iekas VJ-3-31 .11 .46A3 

Outi Laborpraxisloood Labbratory Practice . 

GLP-9oachoiniounolitatNateat of OLF Compliance 

(9amkblaccordiad to S Ills Abo. t Chomikaiionvosots) 

Elm OLP-Inpddion nR Ob-veMmo drr 

ct~P«w~aw tw~ ~ ~ R~ ~'+~°'a"a"'1"'r Dr.a1w WaosaC.n 

MOM wurdo dw'clq~.iK ' 

Faurg.rt nadh Kat.poriftn Arm a F~tfai 

4mo. almvwwo.. ttr. ..t!loOW .weFAd (=M+~ awayWY-OW lar. woaoo VOW-) 

iwagori. z . 

aqMs 

Prorunpoll zar e..twliltrao aar cmdkaogaelm, to*ly.wa.a 

. . . . . . . . 

Katpoda 3 oatapoq i 

~ ~ ~ ~ WWAW&dwROn mut,paMdy dudla 

IgplawMn (in vllro und In vhro) . 

Kaloplia ta eatapory o 

WooMnft oho Too8lkotoplo; . hiodlanlod talodopf; skort Ymo cano~opanl~l~ -low adity pkaM~ao~Or Inlmmlotaotllolo0lot 

Dakun dsr inspokRion , Dato d Impocdon 

iny:&awuwl "W.nwrLrr.n 

30. Aupuat bis 03. 8optotnbsr 2004 on 30 Atpwt un1113 SopErmbor 2004 
 PUBLI'C COPY 

WYl~inrkhq~i O~Andd ~IaA Yn n~bnwn T1w ~Oo~ ~nlawd 1at hd~y b Mdudd In the r~llanw 

wW vAd +@ ' -04 me WCw*I~P~op r~ sad b YmpWMon GmpW 

Elrrrrrrq dr citP.NNnw4Wi obuwweM ewa , 

Ad e.~~ onwwlqs du my.IdoebwietAn ww+ nbied a.ae oe m. +rrpwen nreR k c.. e. qoe*m.a npe ur. 

b"WK aw h dWw ROWxWM do 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 

PUBLIC COPY 

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). 

4"W 20q6' 

Date 

PUBLIC COPY 
p. 20 

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 . Study w.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 of the led material and incubation ... . . .. . .. . ... . . . . . . . . ... ... . . . . ... ... . . .. ... ... . . . . . .. . .... . ... ... . ... .12 8.2 .8. Drbrm(nadon of cog viability (MTT) . .. . .. . . ... . .. . . . . . .. . . .. . ... . . . . . ... ... . .. . . . . ... . ... . . . . .: . ... . .. . ... . .. . . ... . .. . . ... ...12 8.2.7 . Reliability Chedc. . .. . . .. . . .. . ... . . .. . .. . . .. . .. .... . . . . . .. ... . . ... . .. . . .. . .. . . . . . ... . .. . . .. . .. . . .. . .. . .. . ... . .. . .. . . ... ... . .. . . ... . .. . . .. . . .13 

T. RESULTS . . .. . . . . . . . . . .. . ... .. . . . . . . . . . . . . . . . . . . . .. .. . . .. .. . . .. .. .. .... .. .. . . . . . . . . . . . ...... . . . . . . . . . . . . .. .. . .. . . . .. . 14 

7.1 . 

7.2 . 

Cytotoxic indices (viability decresse measured by 14 

Summary of results 14 

8. DISCUSSION AND EVALUATION. . . . . . . . . . . . . . . . . , . .  , . . . . . . . . . . . .14 

9. REFERENCES . . . . . . . . . .. .... .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. .......... .. . . . . . . . . . .. .. .... . . . . . . . . . . . . . . . . .. . .15 
p. 21 

10. AeBREVIATioNS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... .... .. . . . . . . . . ., .. .. . . . . . . . . . .. . . . .... . . . . . . . .1s 

11 . 

11 .1 . 

11 .2. 

APPENDIX.. . .. .. . . . . . . . . . .. . . .... ... ... .... .... . . ..... .. ... . . . . . . . . . . . . . . .. .. .. . . . . . . . . . .. ... . . .. . . . . . . . . . .... .17 

Reiulb .» ... .». ...»..». .... . .»»..» .» ...» ..».. . .... ... .»».»»»».. ....»»».. .»..»».. ...... .... ...... ... ..» .» .»».. ..».»....».». . ...17 

CertMcatloa otValWatioa .» . ..»:» .»..... . ... .... . ..».. ..... ...»....».» . ...». . ... ... ....».»»»»».»». . .» .......»»..»»».».1s 

PUBLIC COPY 
, 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-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 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 of the stu and the methods used have been correctly reported. 

Date: ~ (O Responsible: 
2. SIGNATURES 

Study Director: 

Head of Molecular and Special 

Toxioologyy : 

h tav6 

Date 

9041 f , 06 

ate 

PUBLIC CO Y 
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 of time related cytotoxic effecls the incubation periods were 3 min and 60 min, respectively. By this the study was conducted in accordance with the OECD 431 guideline draft as well as with an EC draft guideline (amending Council Directive 87/548/EEC, B.40 Skin corrosion) using the test item concentrations and incubation periods recommended there. These tests are also related to the revised OECD 404 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 item was 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 item was deterrnined by the assay used. 

PUBLIC COPY 
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 from human 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 aim of these investigations was to test for corrosive properties of the test Item in accordance with existiiQ International guidelines and guideline drafts. 

are retained in the archives specified by 

he storage of a retention sample of the 

France item Is in the responsibility of the 

PU LIC COPY 

~- 6 v 
5. STUDY IDENTIFICATION AND RESPONSIBILITIES 

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: 

.- 

PU LIG COPY 
6. MATERIAL AND METHODS 

The model used for this study has a functional stratum comeum with an underlying 

layer of living cells as recommended by the OECD 431 guideline draft, and EC draft 

amending Council Directive 67/548/EEC, B.40.Skin corrosion . The barrier function of 

the stratum comeum 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 room temperature 

PUBLIC CO 
6.1 .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 EST 1000; 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, 6 well 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 medium and 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 from these 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 item formulation . . 

Test item was 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 item for 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 yellow 3-[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. 

PUBLIC COPY 
6.2 .7 . Reliability Chedc , 

Reliability of the test was confirmed before by interlaboratory Validation (d. Appendix 

11 .2) . . 

PUBLIC COPY 
p. 31 

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 from the information in the Table below the test 

not detected as positive (exceeding the LD50 value 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 LD50 values 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. 

. PUBLIC co 
-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. 

PUBLIC. CO 
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 

PUBLIC CO 
-17- 

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 

' 8 values 

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 ki"814IMS11dilt6tKI4 t*wf-1090 . 

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&D department 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.'h 0":200t1 

soon . 

PUBLIC COPY 

70 
IN VITRO CHROMOSOME ABERRATION TEST 

WITH CHINESE HAMSTER VT! CELLS 

PUBLIC COPY 

]opt) 

StudY Completion Dafis: December 13, 2008 

Page 1 of 46 
PUBLIC COPY 

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 test substanos formulations should be conducted. Suspensions should be 

mixed appropriately prior to administrallonO. 

mber 4, 2008 
CONTENTS 

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. pH and 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 25 
4 

7. References 26 

8. Historical Controls 27 

9. Statement of GLP Compliance 38 

10. Abbreviations used in the Tables 40 

11 . Tables 1-10 41 
p. 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 in writing 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 of the 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 4 hours to concentrations of 2.5, 5 and 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 4 hours 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 blolog icany 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 system for 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 them In 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 Item Is 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 dated August 22, 2006) 

Approved : until December 9, 2006 

Optical appearance' : fine-grained black powder 

Storage : at roorn temperature 

Chemical name 

CAS-No. 

The batch used was analytically examined prior to study Initiation and was approved 

for use for the test period. A stability test in the solvent did not reveal signi8cant degradation of the active ingredient. 

PU LIC COPY 

' don not reflect &emWal oomposidon 
4.1 .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 medium change 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 medium change 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 V79 Calls 

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 from a frozen permanent, 

which was stored In liquid nitrogen, were normally grown In 20 ml medium and 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 from the livers of Arodor 1254 Induced male Sprague Dawley 

rats. The used S9 fraction was derived from the 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 S9 fraction and was kept In refrigerator and used on the same day. 
Cofactor solution per 25 ml S9 mix: 

Sodium phosphate buffer (100 mM; pH 7.4) 15.0 ml 

MgC6 x 8 1-1z0 40" 7 mg 

81 .5 mg KCI 

Glucose-8-phosphate (disodium salt) W.0 mg 

NADP (disodium salt) 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 is M 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 medium of 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~L I 

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 emulsion 
The 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 Interphase were defined as mbft. 

In the main study. cultures with a total InarbatiQ additionally 

and exclusively used to determine the cyboto~ddty 

4.2.6. pH and Osmolality 

Concentrations of up to 100 Ii 

medium In 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 oonflrm the 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 perform a second tog which differa relevantly from the first trial. Therefore, no Independent repeat was needed for the experimental part performed with S9 mix. 

PU 

,;- 9- 3 

L f~; 
The general protocol of the test system was 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 medium per 

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 from the 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"C as described above. After 

4 hours of treatment, the medium was 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 controls. 

4.2.8 . Chromosome Preparations 

The medium was removed from each flask and cogs were removed from the bottom 

of the flask by trypsinizadon and suspended in medium. This medium was transferred to a centrifuge tube and spun for approximately 5 minutes at 700 rpm. The 

supernatant was carefully removed. 

'~- g- 7 
1-2 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 few drops 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. 

U IDs- L i 
Surviving Cella In the Pre-tests 

Experimental Group 

4 Hours Treatment 

without metabolic,8cdvation 

0 

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

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 

"P- 9' 6 
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 

f 

ConcenftWn 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 

Ll 
p. 53 

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. 

PUBLIC CO~i' 
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 100 assessable metaphasea were present on one slide prepared from an individual culture . Therefore, to back-up slide which was generated routinely from every 

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 arm without 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. 

Ll 

a- S" 9 
2. 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 .missing within 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: 

A call 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 comparison 

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. 

A test was considered negative, if there was no such Increase at any time Interval . 

A test was also considered negative, K there 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 significant 
4.8. Assay Acceptance 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 from our laboratory and from published studies. 

4.9. Study Guidelines 

The study was perfomned aoooniing to at least the following guidelines : 

Commission Directive 2000V321EC of 19 May 2000 

Official Journal of the European Communities of 8.8.2000, L 138135- L138/42 

8.10. Mutagenlcity - In vitro Mammalian Chromosome Aberration Test 

OECD Guidelines for Testing 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 Chromosome Aberration 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 of Archives 

Quality Assurance 

Analysts 
s. iteauits 

5.1 . General Remarks 

In the absence and 

posed for 4 hours 

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 C showed also no reduotion In mibosis rate. 

Mibotla Index with 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 index with 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. Chromosome Aberrations 

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 In duding -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 the test 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 4 hours treatment of Chinese hamster V79 cells 

©erbations of 2.5, 5 and 10 Ndm! wa u nd 

of the 

ment 

ment 

addition, after .1 S hours treat- 

.5, 5 and 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 C and cydophosphamide induced dear dastogenic 

effects and demonstrated the sons" of the test system and in the case of cydophosphamide the a*vity of the used S9 mix. 

Based on the results of this 

JIMMOon

sidered 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: A simple 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 from In 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 V79"CoIb 

Hisl Controls of Culluns PnW wd In 1996 

4 Hours Trealnwnt 

Schwa or tiumbK 11rvW IwlBplwns wkh Ywplwu wMb 

IIOc iualhnoa d TlnmIn Abwrdlon, induddp Abarrallorw mdudno 

tmdia mows am" on %) 

Iririrwn 1Adrn mm.,mum 

owon%) 

NiNnrn Nam AAiodmum 

n4m e 1u 0.0 1s 4.e 0 .0 1 .3 4.e 

wdn 1 1t - O.e - - OA - 

1 30 - aa - - CA - 

D4A60 f 1 " 0.0 t0 S.0 0.0 1.6 &0 

" " ~0 OA 1A 4.0 0.0 1 .0 4.0 

momyoU C 10 1 " 20.0 40A M 17.d 40.0 64.0 

mm 6 1 " 110 3A 6.0 1 .0 &0 6.0 

Wabr 1 1" - t.0 - - 1 .8 - 

1 a0 - 1 .0 - - 1 .0 - 

oMw I 1u 1.e sa a.e 1.0 z3 U 

+ " i0 0.0 1 .3 4.0 0A 1 .0 4.0 

L om aphorplwnldr " 1i Y.0 J0.0 'Ws 9.0 a3.0 46.0 

9 UQLIC Copy 
p. 63 

Cyeogwwlks in V""Cdls 

HisboAcal Contr+ols of Culkrn Prnpnd In 1199 

4 Hours Trodnwd 

so llolrwtar Rilla-r 11rwlst AAwkphws via Iwbplsim wah 

IA6t Sub$W= Of T1wIn Abwwlloes kwhWft Ab~emllon~ adudMq 

sudtn MOWS emps on %) 

Yniewre "don mobnwn 

Gps Aw V 

mrinun trUdlmAAmidin 

oiaeo i +i 0.0 O.i s.a 0.0 0,8 a.o 

- e 30 as O.i 1 .5 as ai 1.8 

'an~nydn C " 1i a0 ." 46.0 GU 0.5 40.i GU 

0NS0 i 1" 0.0 1.0 3.6 0.0 0.6 &6 

+ i s0 OA 1.0 as O.i O.i Z6 

~CyCI0pFio~plwlid~~ ! T 18 31 .5 00.i lO.f 31 .11 ,Y.i 00.0 

Cylopnnedas In V7wNb 

Historical Controls of *Own Pnpnrad In 1M 

1" Hours Tnwbnnn! 

0 

Y6t 

soNrwnt a 

labifaW 

Number 

d 

"ludlw 

!lmvwt 

7min 

lfoura 

Wlhplrns wwh 

Awrdloes Insdiv*q 

OWM4%i 

Ai*mIdwm ~an 

YdYpbrw wqb 

Abwrrlon~ mdudq 

aw (ti %) 

db~nw MOM 1 11m., Arn 

na~ 1 fi - 0.5 - - OA - 

" OAAO 7 1i OA 1.0 Z0 10 1 .0 to 

~ Mk"yan e ~ 7 1i M.i 2e.e Vs 10.0 'Ls V.0 

PUBLIC C 
p. 64 

CrEopmwlks in V7""Cdlb 

Htsfortcai Confrols of Cu!lara Piaprrd In Z000 

4 Hours Tnn6nont 

fohrs rt Cr - IlUnbw ilrvut 1Y~I9hws wUh M lo-~s wMh 

Ybt Subshnor d Tim In Aborra0om InoludrV Ab~rdlon~ woolimdft 

obudw Mo~ra Gaps fn %) 

a A~h- n awww 

Seps fn 9F4 

AOninum tiA~dir~ hAroidrn 

W.m e 1s ao 1.s 1 .e 0.0 1.3 1 .5 

e 31 0.0 0.r a.0 0.0 CA 3.0 

omso 10 Is 0.0 1.3 3.0 0.0 1.0 s.0 

" 1o 30 0.e 14 eA CA 1 .8 to 

IiAlomyaYi C 17 1" lam 313 01 .0 11 .0 57.0 61.0 

Wdu " 1" 1.0 3.3 6.0 0.6 Z.0 4.6 

e 30 0.0 1 .3 4A CA 1 .0 43 

DIAlO 11 1" 1 .0 a.0 73 OA ZO 7A 

+ 11 30 0.0 1.0 a0 0:0 1 .0 6.0 

I t CyobpNolplrnidr 19 16 ]0.6 _ l~.0 _ 01.6 ~.0 49.5 H.b 

CyboqerANbs in V7!"Caib 

Hislborlwi Controls of Culbrns Fnprrd In 2000 

1Is NOW* Trombnom 

N 

wbt 

sa1wM a 

lubohnom 

I*rnb~r 

at 

ftmom 

1lrvnt 

TWO In 

Noun 

UoIopwes wlTb 

Aborraftr kmUadFp 

aw on %~ 

I*reue I I I Mrd~~n 

Ywaplrws wllb 

Ab~naaom oxdiWY 

OW (in xa 

AOnYnu n MIdim MmMre 

waar e 10 0.0 to L0 0.0 1,0 a.0 

" omeo 0 to as IA aA CA to as 

( mrornyoin c ~ 1e 1u ao 2e.o a.o 8.5 aco 4a.o 

PUBLI 

~- 7 9 

C 
CyEoPnotics In v74.Cdls 

Hislorical Conbols ol'Culhrns PnW nd !n Z001 

4 Hours Trftbnwd 

Is "olwit or Number ihrvest INsdpirns wilh Ilslsphrss wMh 

Ybe suesEmo. oa TIm.1n Ae.eaean inew~dlno llesrrseoasmaudlno 

ftwke NOUN Caps On %) 00110011%) 

Wdw 0 1s OA 0.0 20 0.0 0.0 t .0 

4 30 OA 1 .3 1.5 as 1.3 1 .0 

OM 11 1s 1.0 1 .6 4.0 Os 1 .0 4.0 

" 11 so as 1 .0 4s as 1.0 4s 

FOb~11yqY~ c 10 1s 16s 41 .0 l6.6 72.6 40 .0 !0.0 

Wdrr 4 Is 1 .0 M 4.0 1 .0 &s 4.0 

4 30 a0 Os 1.0 0.6 0.6 1.0 

D1AS0 11 1s 0.6 2A 0.0 O.s Z.0 5.0 

a 1f SO 0.0 1 .0 43 0.0 1 .0 4.1 

I , Cyclfpho~ph~nidol 1s T is r =s 40A 0l.0 MA 45.0 l0.0 

Cyleenadas in V79-CoIIa 

Hlstor10a1 Conh+0ls of Cultures Pnpvad In Z001 

Ill, Hours Tnm*nwit 

p 

Ilk 

SW- it a 

subsfsnos 

lpwibsr 

of 

Shmdlss 

llsrwd 

Time M 

Hours 

lbtiplwsss wrMh 

Abunslloss Igh, A @ 

am on %I 

Mni~w~ ~Idri AYa~Ynun 

Wlspirsss wwh 

AbsrrWoes =&WIno 

Sells on w 

I~Ynum AA~r~ t~A~odrtun 

wow 4 1s 0.0 0.3 1 .e 0.0 O.a 1.e 

" oMeo s is 0.e ss ss 0.s :0 ss 

~ Mrmnyanc 12 1s 15.6 s1 .s 4a.o 1ao tsA 4a 

PU LIC CO 

3ov 
31 

Cytorneda !r V7!"Cdb 

Htrtorkol CanMb etCatarno PreMnd in ?A03 

4 Hom 'Prettmeot 

N solwik a NumW Ilrrwt Y~Miplr~s wMh UWtlphrn wub 

flbc W6d4na of 71m In Abwr~flau kwkde0 Iib~era0orr wxluWp 

aeudlw No" omps (n 'Xl COPS on %) 

wd.r 7 1s 0 .0 as aA 0.0 o.e a.o 

e 30 0.0 :0 4.0 0.0 1 .4 4.0 

DIiA90 1 " 1" OA 1.3 4.0 0.0 1.0 &0 

" 1" !0 0.0 1A &0 0.0 1.0 a.0 

Elhn01 1 1 " - 2.0 - - 4.0 - 

1 30 - 20 - - Z.0 - 

Ill,aryeJn C as 1" 30A 47A !4A 34.5 40.0 !20 

war . I 1s 0.0 1.6 ao 0.0 1 .4 to 

6 30 04 10 4.0 OA 10 4.0 

DM00 1" 1s OA 1 ." 4.f OA 1A 4.6 

+ lo 30 0.0 is 4.0 0.0 1.e 4.0 

Oh" 1 u - 4,e - _ 4,0 - 

1 30 - U - - o,e - 

~ CyeloOtio~plrnid~ 2Q 1" 3R0 4'1A !IA Us aQ.O ~.6 

PUb"LIC C 
Cybpne8p 1s VT!"CeW 

Hwarkal C=troM o[ Cwtvrw h+vpwwia 2m 

1sHeors 1rabmt 

W 

wk 

solwnt or 

Subdr" 

Um ON, 

of 

alsllw 

Il~rw~t 

Tlns in 

"am 

me"hws whh 

Abortitais tidudMq 

ftppn w 

tAnY,m OA~d~n Ywir~nr 

Alofophrw wwh 

Ab~allorr roodudbq 

ft" on %) 

Ai*,m IAl~dr~ 1Awinum 

wow 0 1" 0.0 110 AO 0.0 0.0 3.0 

" WOO 16 ti 0.0 td GA 0.0 ZO 6"d 

on" 1 1" - 1A - - 1 .0 - 

I WIN qck ,C 22 ,. 1" 3 .0 ,..0 1&0 m ,.s 

PU LIU C 
Cybogwwflas in V11f-Cdfs 

HbtorIcN Cordrols of Culluns Pnpund in 2003 

4 Hours Tnnbeme! 

"aNwd or Numbw llor"4t Y~I~plwowe YsImplwss wllh 

Albc sufmWma of T1ne In AbomHons UMhWkV Ab~rnllons =dWft 

ftmft "Mrs on %) 

wiiwum MOM Mo*nm 

Ow on %) 

A~iiYnun Medwn Mmnrn 

waw. 10 1. as to GA 0.e za e.o 

8 30 oA 2A s.0 MO 10 4.e 

- omeo 14 u 1 .0 ao GA 0.0 &0 to 

14 30 1 .0 13 7.0 0.e 2.0 74 

awomyorn c 24 1o a1A 4" eoA SoA 31Lo esA 

wow 0 10 OA 11 0.6 0.0 1A 5.6 

0 30 0.0 20 a0 OA 1A 4.6 

am 14 10 OA S.0 0.0 OA 0.0 0.0 

14 30 OA IA 7.0 0.4 1.0 7.0 

07ol Oplg~hnlld~ U 9 310 40.0 06.0 51 .0 40.0 N.0 

CyloOwwNas In Vff"CNIs 

Hlabork.nl Contruls of Whores Pnpend In 2003 

1" How* Trnn4nmnt 

!~ solwnt or Ilwnber NN w ~t Ilimphr"wMh 1lwtihplrlles wNb 

Y6c dubshrn of 7froIn Abwnllau inewnp AbKrooes rocdWnq 

Skidiw NOW* GO" on %) Omps fn %) 

w4.r s 1a as a.0 aI 0.s aa as 

" oMeo s 1o to a.0 4A 0.s 3.4 4.s 

Moa+yanc 17 1e u.o 31 .0 eos 1e.0 Al 47.s 

UBLIC 

305 
cymowwdm in V19-CAM 

Hhtorksl Controls o1 Cullures P4+spnnd in 2004 

4 Hours TruNmnt 

!~ so-~t ar 1Urwibsr Mrwst Nabob"" wo 

Aft subsOnos or TIm In lls.n.usns lududhq Absrradons emkde, 

saa"Ales Nas Ss" on %N aa th %1 

waW 2 Is 1.6 4.6 ?.e 1.5 s1 6.8 

2 30 1.5 13 10 1 .5 13 3.o 

orao Is u OA ta 7.s OA ta es 

Is 30 0.5 Is 4.0 OA 1 .5 4.0 

" Erred ~ u - as - - 2e - 

ao - 3.0 - - s.o - 

Nrawyanc ~s u 2" 4Ls KO As ass e~s 

w.i.r z Is o.e 4s Ss as 4.0 zs 

2 30 to " 5.0 2.0 s0 e.o 

ONso » u 0.0 as 4.0 o.o ss GA 

+ +e ao o.s as ao as as LO 

EIWa I u - s.e - - 4s - 

30 - s.0 - - 2.0 - 

1 q~ao~o~o~rnr1. 'o 18 W.0 M.0 s1.e 31 .0 40s 0.0 

U LIC COPY 
Cybwmnlics In m"Cdls 

Hisforlcd Controb of Cularns A *M In 2004 

18 Ham TnwbmM 

a 

OAk 

sdvwt er 

sub~Yno~ 

Mwrm 

af 

s,wv.. 

Nrw.e 

The . In 

Hem 

Y.dpw.. wwh 

ft rn.Non.6iduabo 

a" An %1 

r.l~pnr.. wwn 

I1bu*rloas.RaudMg 

ft" (ti xf 

wwr 3 u to 1 .0 s.e la 1 .0 as 

" omwo Is +u ae a.o 7.s o.o :.e ae 

err~a t Is - a.o 

+ mbeydn c 19 u aae AS 4e.e 0.5 

PUBLIC COF`~ 
Cyogwwllcs In V7'4-Cdls 

His§orkd Controls d Cultures Prepared in 2005 

4 How* Tnalrnnnt 

Solwne or pwnbw Nrvwd Wimphrsewllh IIWhplrw wwh 

Mk subdhnos of TIw In Ab~nrflon~ Indudlno Ab~naUon~ =dudlno 

skmft WIN rn Gap fn xl 

Mai- III m - 

GWM %1 

nmm maw nm - ~ 

w.1v 2 M 0.0 o.o 0.0 0.0 o.o 0.0 

2 30 al oa 

1

.0 

0

.e as + .0 

oaiso 2+ u 0.e 2.0 e.e 0.e 2.0 4.0 

19 30 0.0 so e.0 o.o 1 .e 4.0 

- eh" I Is - a.o - - 2A - 

30 - 1.a - - 1 .3 - 

EWE I Is - 11 .0 - _ 10.0 - 

30 - 5.0 - - e.o - 

hognaar, c as u - 15.0 41.0 M Is.o 4s.o 710 

w.i . s u Ze 4.e e.e to 4.e Ls 

2 30 1.0 is 20 1.0 1 .e 10 

oNso 21 u IA a.o s.1 1 .e s.o e.o 

+ Is 30 o.e :e es 0.e 10 e.e 

~rna ~ w - 4.s - - 4,8 _ 

ao - i.o - - Is - 

two > >s - in - - :o - 

30 - se - - 3.0 - 

as ou as -a-0, 

PUBLIC COPY 
Cykv.w .em in v79-c.n. 

Mbboftal Controb of Culaurs P.*W nd (n 2006 

1i Meum Tr"bn&nt 

I~s 

Solwnt or 

submlua 

Nueibw 

of 

oWes 

1lrwrt 

T1m b1 

Hoan 

1U1eprssewqA 

Ab~ndloe~ - A 0 

Go" on !y 

Ilaimplrses wo 

Abwrarom aoocludlng 

wps on %) 

1Ndw 2 10 1.0 1 .3 1 .5 1.0 1 .0 1A 

- DAO)Q 1" 1" OA Z.! "A to 20 6.8 

EM 1 f0 - t" - - 5.6 - 

r AA101yoYi C 1i 1" 120 31.0 10.0 12.0 30.8 16.0 

PUBLIC COPY 
p. 73 

. 9bbflleflt & GLP COfl'ipft11f.y 

i0r Umwoh uod N La.dwubchdt uod 

Varbeuc6eempaz 

aa Lmae.N«+a~a~-w 

l1~~ilrM Or~ Alrl" sYLf9l .Il,lir 

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maE : mulWe abeRadons with exchange 

cd : chromosome disintegmtlon 
p. 76 

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. p. 82 

SALMONELLAIMICROSOME TEST 

PLATE INCORPORATION AND PREINCUBATION METHOD 

Study Completion Date: December 15, 2008 

Page 1 of 56 

PUBLIC COPY 
2 

OLP Compliance 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 item in 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 COPY 
CONTENTS 

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 (pKM 101) 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 

PUBLIC COPY 
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 

PUBLIC COPY 
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 PREPARATION OF 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 rpmdWf the study and the methods used have been correctly reported. 

Date: Ice_ Aft, Aft` 

3~- ~ 
1 . Signature 

Study Director. December 15, 2008 

Date 

PUBLIC COPY 
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 typhimurtum LT2 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 COPY 
monellalMicrosome 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). 

PUBLIC COPY 
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 item is in the responsibility of the sponPUBLIC COPY 
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 composition 
4.1 .2 . Positive Controls 

Sodium azide (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 direct acting 

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 direct acting 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 COPY 
monellalMicrosome 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 G 46, and TA 100 additionally contains the plasmid pKM 101 . 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)- f 
aImoneIla/Microsome Test 

13 

TA 1538, which differs from TA 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 Prof. 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°C for approximately 24 hours. Samples were taken from individual 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. New samples 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 from these 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 DMSO to 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 UV sensitivity (uvrB) where approprIate. .The crystal-violet and UV sensitivity 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 

32-7 
Whenever new stock 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 (pKM 101) 

A spedal 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. 

PUBLIC COPY 
a mone e Test 

15 

4.2 .4.4. Crystal-Violet Sensitivity (deep rough) 

A quantity of 0.1 ml was taken from the samples of individual stocks and spread onto 

nutrient agar, using four plates per strain. After a few minutes, 51ter papers, to which 

10 Irl 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 aluminum foil 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 nm at 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 COPY 
4.3. S9 Mix 

S9 mix was used to simulate the mammalian metabolism of the test substance. It 

was made from the 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 S9 fraction) 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 Ice and used only on the same day. 

Seventy ml of cofactor solution are composed as follows: 

MgC6 x 8 H20 182.6 mg 

KCI 246.0 mg 

Glucose-8-phosphate, disodium salt 179.1 mg 

NADP, disodium salt 315.0 mg 

Phosphate buffer 100.0 mm 

S9 mix consists of this cofactor solution, S9 fracdon and, If needed, 0.15 M KCI. The 

amount of S9 fraction In S9 mix is Indicated In the tables In percent. The SO mix 

comprised the amount of S9 fraction (x°16) Indicated in the tables, 70% cofactor soiutlon and (30-x)°X, 0.15 M KCI. The S9 fraction was derived from the preparation dated 

August 22, 2000. (protein content 37.6 tng per ml). 

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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 48 hours 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 

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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. 

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Rtrwau+. 

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The doses for the first trial were routinely determined on-the basis of a standard protocol: if not limited by solubility 5000 jig 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 the first experiment . 

The independent repeat was performed as preincubaflon In a water bath at 37°C for 

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 

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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. . 

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19 

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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