p. 1 Page 13 of 17 Study number Test Item CAS number : RESULTS Test Itemconcentration [m0/L] : Control Abiotic parameters 011 24 h 48 h 72 h % h T tun °C 22.1 22.6 22.6 22.5 22 .3 Oxygen [m82] 8.4 7.9 8 .0 8

.2 8

.1 Oxygen [%

saturation] 96 92 92 95 95 pHvalue 8 .0 7.7 7.7 7 .8 7

.8 Mortallty for zebra fish (initial population: 10) 2 h 24 h 48 h 72 h % h Absolute 0 0 0 0 0 Cumulative 0 0 0 0 0 Cumulative [%] 0 0 0 0 0 Comments : none 

# PUBLIC COPYp. 2 Page 14 of 17 Study number Test Item CAS number :

Teat itemconcentration [mg/L] : RESULTS 100 Abiotic parameters 0h 24 h 48 h 72 h % h T emperature °C 23.1 22.6 22.5 22.5 22.2 Oxygen [mg/L] 8 .3 8

.0 8

.1 8

.3 8.2 Oxygen [%

saturation] 98 93 94 96 95 pH value 8 .1 7

.8 7.8 7

.9 7.8 Mortnlity for zebra 8sh (initial population: 10) 2 h 24 h 48 h 72 h % h Absolute 0 0 0 0 0 Cumulative 0 0 0 0 0 Cumulative [%] 0 0 0 0 0 Comments: none 

# r &UBLIC 

# COPYStudy namber

Test item CAS number : RESULTS Analysis ofthe mortality data gives the following results: Time LC 50 

### [N tIng/M 2 > 100 24 > 100 48 > 100 72 > 100 96 > 100 No toxic effects against fish at 100 mg/L. p. 3 Page 15 of 17

The test itemwas insoluble in water. After addition to water, it formed small lumps that did not disperse by stirring. Treatment in an ultrasonic bath resulted in black colouration of the aquariumcontent, indicating very fine dispersal of the test item In the first hours of the test, fine flocs oftest itemformed and partly settled on the bottom ofthe test vessel . The results are expressed in terms of nominal concentrations.



# PUBLIC COPYp. 4 Page 16 of 17 Study number I Test Item CAS number : ARCHIVATION OF DOCUMENTS ANDTEST ARTICLES Study plans, raw

data and study reports are archived at the GLParchives of the test facility. Test articles are archived The sponsor must ensure that information submitted about the test item(identity testing or information about physical and chennical properties) is complete and that it complies with the principles of Good Laboratory Practice. The cus le of the test itemused where sampling has not been performed 

# PUBLIC COPYp. 5 Page 17 of 17 Study number Test item CAS number 4

: . 1 

# 0

_ 

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WMYY+ wWer+w~errprt~" t1RU~hdrYIa1 qOdArI " doe 1~. q " its ~p1~~0 " t a~ 14w " 1" l~ilriiiroi a~ 1! ad7t ,wp .y . OU ~, CIdaU" rT006 oNbOr7006 or~r,~r~+r " wwr rw r .,w~wnrww~ .r .w~w~r~rwwrnr .r ~k~~wwwr

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. ~~.+ rwra~rr~ww+wawr~wwr .r .rrwn wrr wrwr ~~ do au .wrri .0rwrew~av . rrawrMp. 6 page 1 of 41 

# PUBLIC COPY

ALGA, GROWTH INHIBITION TEST 

### Author :

Sponsor : Testing facility:page 2 of41 Study number Test Item CAS number : -

page GLP DECLARATION CONTENTS

QUALITY ASSURANCE STATEMENT 5 RESPONSIBLE PERSONNEL 6 SUNIIVIARY 7-8 INTRODUCTION 9 EXPERIIVIENTAL PROCEDURE 10 MATERIALS ANDMETHODS 11

- TEsT ITEM - TEsT srEcms ~Z r `~, t~ . 11 12 13 13-14 15 16 - PRE-TREATMENT OF THE TEST ITEM - ExposuRp coNDrnoNs

- EXPREssIONOF BIOLOOICAL RE3ULTs

- APPLMD SOPS RESULTS 17-30

, ~ ; ~ ~~ TES :: . .

- BvF-- 31 ARCHIVATION OF DOCUMENTS AND TARTICLE , 

# PUBLIC COPYStudy number ~ Test Item : CAS number : CONTENTS (continued) page 3 of41

page 

## FIGURE 1 : CONCLNTRATiON-EFFECT CURVE (EeCVALUES) 32 FIGURE 2

: CONCENTRATION-EFFECT CURVE (ECVALUES) 33 FIGURE 3 - 4

: GROWTH CURVES 34-35 ANNEX 1 : NUTRIENT MEDIUM PRE-CULTURES ANDTEST CULTURES 36 ANNEX 2

: EQUATIONS 37-38 ANNEX 3 WILLIAMS MULTIPLE SEQUENTIAL T-TEST PROCEDURE 39-40 GLP CERTIFICATE 41 

# PUBLIC COPYStudy number ;/ Test item ~

. CAS number : - page 4 of 41

GLP DECLARATION This study was performed in accordance with the principles of Good Laboratory Practice (GLP) as specified in Annex I of Law on Hazardous Substances (German Chemicals Law - according to the respective valid issue) .

Date / Signature Study director 

# PUBLIC COPYpage 5 of 41 Study number . Test item CAS number : QUALITY ASSURANCE STATEMENT STUDY NUMBER TEST II'EM TITLE : Alga, Growth Inhibition Test The Quality Assurance Unit inspected the performing of phases of this study and examined the study report . The head of the test facility was informed on the results ofthese inspections at the dates mentioned below . Date of p

lan i nspection: Date of S report: specification study plan 2007-02-02 2007-02-02 Date of A i nspect ion: Date of S report: process-based inspection SOP 00001 2006-08-23 2006-08-23

Date of report i nspection: Date of report i nspec tion: 

### 0?00'3-o5- -If S ?ego~ -or-'/SThe results of this study have been examined on the basis of the valid SOPs and we confirm that, to the best of our knowledge, the raw data have been stated correctly. Date: 0200 ~ -OS- 95' QuaHty Assurance Unit :p. 11 Study dumber " Teat item CAS number : -- RESPONSIBLE PERSONNEL page 6 of 41

Date / Signature



# PUBLIC COPYStudy number Test Item CAS number : page 7 of 41

SUMMARY A study was performed to assess adverse effects pn the growth (_ - - -- -- - increase in cell density) and the growth rate (= rate of increase in cell density with time) of the planktonic freshwater algal species Desmodermu .4 subspicatmover several generations. The study was conducted in accordance with EEC Methods for Determination of Ecotoxicity Annex to Directive 92/69/EEC (O .J. No. L383A, 29

.12.92) Part C, Method 3 'Algal inhibition test' which is in most parts equivalent to the OECD Guideline for Testing of Chemicals No . 201 'Alga, Growth Inhibition Test' . Exponentially growing algal cells were exposed for a period of 72 concentrations, nominally 0 .9, 1 .9, 4 .3, 9 .4, 20.7, 45.5 and 100 mg/L dispersed in water. . " The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth (index b) and growth rate (index r), relative to control cultures grown under identical conditions. Growth and growth rates were used to calculate a No Observed Effect Concentration and a Lowest Observed Effect Concentration according to WILLIAMS Multiple Sequential West Procedure. The following values were determined: Results [mg/LJ : 95% confidence limits [mg/L] EbC 50 (0-72 h) : 23 12-52 EbC 10 (0-72 h) : 2.3 0 .14-5.6 ErC 50 (0-72 h) :134 72-872 ErC 10 (0-72 h) : 8 .9 0

.76-18 NOEC [b] : 0

.9 NOEC [r] : 0 .9 Additionally, a filtered suspension of nominally 100 mg/L was tested, and resulted in 10 .0% inhibition (growth rate, 72 h) compared to the control .



# PUBLIC COPYp. 13

Study number Teat item page 8 of 41 CAS number : SUMMARY The test item

was insoluble in water . Treatment in an ultrasonic bath (S min .) resulted in black colouration of the flask content, indicating fine dispersal of the test item . After treatment on a magnetic stirrer for 1 h, the suspension was tested without filtration. The results are expressed in terms of nominal concentrations. To reduce shading effects of the coloured test substance, the test was carried out with strong illumination (80 -120 pE) and with a reduced test volume of 25 mL in 300 mL Erlennmeyer flasks. 

# PUBLIC COPYp. 14 page 9 of 41 Study number " Test Item CAS number : - INTRODUCTION and the results obtained during a study This report contains a = Vi m" to investigate adverse the growth and the growth rate of a population of the planlctanic water algal species Desmodes»nwsubspicatrrs. In the present test, adverse effects are expressed as those effective concentrations which reduce growth (index b) and growth rate (index r) by 50% (ErC 50, EC 50) within a continuous 72 hour period of exposure. In those cases where the slope of the concentration/percentage response curve is too steep t3-permit calculation of the EC 50, this value is estimated graphically

. in items of minor toxicity (or low aqueous solubility) a limit test is performed at 100 mg/L (or at the limit of water solubility) in order to demonstrate that the EC 50 is greater than this concentration. In addition to the EC 50 value, NOEGLOEC is calculated based on growth [b] and growth rate [r], respectively . The study was commissioned 2007-02-02 and 2007-05-04 ( d undertaken between 2-03 to 2007-05-04) . 

# PUBLIC COPYStudy number Test Item CAS number : -- EXPERIMENTAL PROCEDURE page 10 of 41

The method described in the Council Directive 92/69/EEC (O .J. No . L383A, 29

.12 .92) Part C, Method 3 'Algal inhibition tesN assesses adverse effects (inhibition of growth and growth rate) of various concentrations of a test item to a unicellular planktonic freshwater algal species. The purpose ofthis method is to determine those concentrations which cause a 50% adverse effect (= EC 50) or, if conducted as a limit test, to determine the adverse effects at a maximum

test concentration of 100 mg/L or at the limit ofwater solubility, respectively . All effect data are expressed on the basis ofgrowth [b] and growth rate [r] . A range-finding test may precede the main test. It provides information about the range of concentrations to be used in the main test. In the main test, the algae are exposed to the test item added to water at a range of concentrations for a period of 72 hours. Defined concentrations of me test itemwill lead to a certain inhibition ofalgal growth and growth rate at the end of the 72 hour study period. Cell densities are recorded at 24-hour intervals. The 72 hour EC 50 (based on both, growth [b] and growth rate [r], is calculated or read from the concentration/percentage response curve . During the test a temperature range of 21- 25 °C is to be maintained in the test vessels. The pHis measured at the beginning of the test and at 72 hours. The cell density in the control cultures should increase by a factor of at least 16 within 72 hours. The concentration of the test itemshall ideally be maintained to within 80%of the initial concentration throughout the study . In order to avoid an impairment of the test system, an additional replicate is used for pH measurement at the beginning of the test (control : replicate VII ; test concentrations : replicate IV) . PH measurement is performed using the total volume replicates I-II .



# PUBLIC COPYStudy nnmber

Test Item .

Iq CAS number : --

Teat Item CAS number CASpreferred name Chemical name Article number Batch Empirical formula MATERIAL ANDMETHODS

. page 11 of 41

Structural formula . --- Molecular weight . --- Purity : 95.8% Major impurities . ---- Water solubility : insoluble (according to data of the sponsor) Vapour pressure . --- Chemical stability in water and light Stability of test concentration/s during exposure : 

# PUBLIC COPYStudy number page 12 of 41

Test item CAS number : MATERIALS AND METHODS Test snecies Name : Desnrodesmas subspicatus Source : Non-axenic strain of the test species obtained from The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Gbttingen (Germany) Maintenance of stock cultures . Exponentially-growing stock cultures are maintained in the test facility under constant temperature conditions (23 +/- 2°C) at a light intensity in the range 60 - 120 pE. x m7 2 x s" (measured in the range 400 to 700 nn using a spherical quantum flux meter) . The nutrient medium(according to BRINGMANN 8t KOHN (1977) is renewed once a week. Cell density measuroments are made using a microcell counter. Preparation of pre-cultures : Pre-cultures are set up three days before the start of a test. They are grown under identical exposure conditions as the stock cultures, except fromthe use of a different nutrient medium(annex 1) . Test cultures The algal inocula for a test are taken from an exponentially-growing pre-culture and are mixed with the nutrient medium(annex 1) to make up to a final cell density of about 5000 cells per millilitre in the test medium . 

# PUBLIC COPYStudy numbe

Test Item CAS number MATERIALS AND METHODS 

### r mdmm en

### t o 

### ft h

### e tes

### t i tem page 13 of 41

A stock "solution" was prepared to give the desired series of test concentrations. To achieve this 125

.1 mg of the test itemwere added to 1 litre of deionisiered water and treated for S min. in an ultrasonic bath and afterwards stirred for 1 h on a magnetic stirrer. Undissolved particles of the test itemwere not removed. The pH was adjusted to pH 8 .3. Of this solution, 20 mL were taken and 2 .5 mL of concentrated mineral medium and 2 .5 mL of algal inoculum

were added

. To produce an additional test concentration of nominally 100 mg/L with filtration, 111 .2 mg of the test item was added to 1000 mL ofalgal medium, treated for 1 h in an ultrasonic bath and afterwards stirred for 24 h on a magnetic stiaer. Finally undissolved particles of the test itemwere removed by filtration using a folded filter of pore size 7-12 pm

. The pH was measured to be 8 .5 . The suspension was passed through a folded filter, resulting in a clear filtrate. Of this filtrate, 22 .5 mL were added to 2 .5 mL ofalga inoculum

. Exposure conditi ons Test vessels : 300 mL Erlenmeyer flasks with cotton stoppers Culturing apparatus : Light chamber in which a temperature in the range 21 °C to 25 °C can be maintained at +/- 2 °C, and continuous uniform illumination is provided in the spectral range 400to 700 nm . Magnetic stirrers kept the algae in suspension Light intensity At the average of the test solutions, a light intensity in the range 80 to 120 EtE. x m7 2 x 9 ", or an equivalent range of 6000to 8000 lx, is recommended to use . Cell density measurements : Cell densities are measured in a microcell counter or, alternatively, are determined by means of a microscopic counting chamber. 

# PUBLIC COPYStudy nnmbel Test item CAS number : MATERIALS AND METHODS Eznoanre conditions page 14 of 41

Experimental design : 8 test concentrations plus 1 control 3 replicates per concentration, 6 replicates per control initial cell density in the test cultures approximately 5000 cells permillilitre Test concentration/s (nominal) : 0 .9, 1 .9, 4

.3, 9 .4, 20 .7, 45 .5 and 100 mg/L; also 100mg/L filtered solution Method ofadministration : stock "solution" Duration ofexposure : 72 hours Criteria ofeffects : The criteria of adverse effects used in this study were the item induced inhibition of growth [b] and growth rate [r], respectively, of the algal population. 

# PUBLIC COPYStudy namber page 15 of 41 Test Item CAS number

MATERIALS ANDMETHODS Eznression of bioloai alres 1ts

"

"

" Cell density measurements in the test and control cultures are tabulated according to the concentration oftest itemand the time of measurement. " Growth curves are plotted for each test concentration and control . The area under the growth curve [b] is calculated for each test culture using equation [1] in annex Z. The growth rate [r] is calculated for each test culture using equation [3] in annex 2 . The percentage inhibition of both, growth [b] and growth rate [r], is calculated for each test concentration using equations [2] and [4] (annex 2) . " If possible, EC 50 and EC 10 values for both, growth [b] and growth rate [r], are calculated by probit analysis . If a limit test is performed it has to be demonstrated that the EC SO is greater than this concentration. If possible, NOW

and LOEC of both, growth [b] and growth rate [r], are determined by a multisample comparison (according to Williams Multiple Sequential t-test Procedure) (annex 3) 

# PUBLIC COPYStudy nnmbe CAS number : page 16 of 41 Test Item

MATERIALS AND METHODS ADDIi ed SOPs SOP number Function Validity 00093 V .1 Balances Feb. 01, 2006 2030-6600401-96 D Direct weighing Nov . 29,1996 2030-1110102-02 D pHmeasurement June 1, 2002 2030-7000806-01 D Algal growth inhibition test Sept . 1, 2001 Deviations : No chemical analysis was performed, The cell density at test start was 5000 cells/mL. 

# PUBLIC COPYStudy nnm Test item ~ CAS number : RESULTS Teat item

concentration [mg/L] : Control page 17 of 41

eplicate Cell density IceWmL]

(initial cell density = 5000 cells/mL) 24 h 48 h 72 h Growth (b) [~~of bio°uass] Growth

rate (r) 1 30000 130000 400000 347500 1 .46 II 23333 170000 580000 470833 1 .58 III 40000 130000 680000 497500 1 .64 IV 20000 100000 423333 319167 1 .48 V 40000 170000 740000 567500 1 .67

40000 110000 706667 490833 1 .65 0 32222 135000 588333 448889 1 .59 Growth (b) Area under growth curve (0) : 448889 Percentage inhibition ofcell growth . 0 .0 Growth rate (r) Specific growth rate (0) . 1 .59 Percentage inhibition of cell growth rate . 0 .0 pHvalues Analysi1 

# 1 Oh 8.2 

# I 72 h 75 

# I PUBLIC COPY- page 18 of 41 Study number Test Item CAS number 

### RESULTS* Test itemconcentration [mg/L] : 0 .9 Replicate Cell density [cdWmL]

(initial cell density = 5000 cells/mL) 24 h 48 h 72 h Growth (b) [~~l of biomasr Growth

~~ (r) 1 26667 106667 570000 405833 1 .58 11 30000 133333 600000 450833 1 .60 III 26667 156667 643333 492500 1 .62 0 27778 132222 604444 449722 1 .60 Growth (b) Area under growth curve (0) : 449722 Percentage increase ofcell growth . -0 .2 Growth rate (r) Specific growth rate (0) . 1 .60 Percentage increase of cell growth rate . -0 .6 pHvalues Analysis I Oh 8 .2 j 72 h

8.5 

# I PUBLIC COPYStudy num"'---J Test Item CAS nnmber : RESULTS Test itemconcentration [mg/L] : 1,9 page 19of 41

Replicate Cell density [ceW/mL]

(initiaL cell density = 5000 cells/mL) 24 h 48 h 72 h Growth (b) [integmi of biomass] Growth

rate (r) I 23333 133333 336667 312500 1 .40

40000 183333 343333 382500 1 .41 26667 103333 396667 315833 1,46 0 30000 140000 358889 336944 1 .42 Growth (b) Area under growth curve (0) : 336944 Percentage inhibition of cell growth . 24.9 Growth rate (r) Specific growth rate (0)

. 1 .42 Percentage inhibition ofcell growth rate . 10.4 pHvalues 

## AnItysi . 

# I 0b

8 .1 

# l I 72 h

8.4 

# I PUBLIC COPYStudy number l . Item " CAS number : - RESULTS Teat item concentration [mg/L] : 4 .3 page 20 of 41

Cell density [celWmL] Growth (b) (initial cell density = 5000cells/mL) [~~of Growth Replicate 24 h 48 h 72 h biomaa~] ~~ (r) 1 36667 96667 486667 364167 1 .53 11 36667 193333 373333 404167 1 .44

36667 116667 473333 377500 1 .52 0 36667 135556 444444 381944 1 .50 Growth (b) Area under growth curve (0) . 381944 Percentage inhibition of cell growth . 14 .9 Growth rate (r) Specific growth rate (0) . 1 .50 Percentage inhibition of cell growth rate . 5.9 pHvaluea Analysis 

# I oe 8.0 

# I I 72 h

72 

## 0 

# I PUBLIC COPYStudy number Test Item CAS number :

Test item

concentration [mg/L] : 9 .4 .



# I Cell density [ce1WmL] Growth (b) (initial cell density = 5000 cells/mL) [~~of Growth ~~ (r) Replicate 24 h 48 h 72 h blomaae I 23333 153333 340000 334167 1 .41 II 30000 193333 366667 394167 1 .43 III 30000 96667 266667 247500 1 .33 0 27778 147778 324444 325278 1 .39 Growth (b) Area under growth curve (0) . 325278 Percentage inhibition of cell growth . 27.5 RESULTS

Growth rate (r) Specific growth rate (0) Percentage inhibition ofcell growth rate 

# F pH valum 

# I h 8 .o Analysis 

# I page 21 of 41

1 .39 12.5 72 h 72 

## 7 

# I PUBLIC COPY-- page 22 of 41

Study nnmbeli~ Test Item CAS number : RESULTS Teat itemconcentration [m0/L] : 20 .7 Cell density [celle/mL] Growth (b) E (initial cell density = 5000 cells/mL) [Integral of Growth rate (r) Repflcate 24 h 48 h 72 h biomaee] 1 26667 83333 403333 299167 1 .46 II 30000 93333 426667 324167 1 .48 III 30000 63333 283333 222500 1 .35 0 28889 80000 371111 281944 1 .44 Growth (b) Area under growth curve (0) . 281944 Percentage inhibition of cell growth . 37.2 Growth rate (r) Specific growth rate (0) Percentage inhibition of cell growth rate pHvalues Analysis 

# I Oh _ 80 1 .44

9.7 72 h 

## 8.1 

# I PUBLIC COPYStudy nnmbeAl Test item CAS number : RESULTS Test itemconcentration [mg/L] : 45 .5 page 23 of 41

Cell density [cells/mL] Growth (b) (initial cell density = 5000 cells/mL) [Integral of Growth Replicate 24 h 48 h 72 h biomass] rate (r) 1 30000 40000 83333 99167 0 .94 11 26667 53333 196667 165833 1 .22 lII 13333 50000 60000 80833 0 .83 0 23333 47778 113333 115278 1 .04 Growth (b) Area under growth curve (Q1) . 115278 Percentage inhibition ofcell growth . 74.3 Growth rate (r) Specific growth rate (0) Percentage inhibition ofcell growth rate pHvalue; Analyab 

# I Oh 8.1 I 72 b 70 

# U V I 

# Ow"'LIC 

# CO

1 .04 34 .5 

# IStudy numbe Test Item CAS number RESULTS Teat itemconcentration [mg/L] : 100 page 24 of41

. Replicate Cell density [celle/mLI

(initial cell density = 5000 cells/mL) 24 h 48 h 72 h Growth (b) [integral of biomrus] Growth

rate (r) 1 40000 53333 73333 117500 0 .90 11 16667 33333 60000 67500 0 .83 16667 40000 86667 87500 0 .95 0 24444 42222 73333 90833 0 .90 Growth (b) Area under growth curve (0) . 90833 Percentage inhibition of cell growth . 79 .8 Growth rate (r) Specific growth rate (0) . 0 .90 Percentage inhibition ofcell growth rate . 43.7 pHvalues Analysis 

# I 8.1 

# I I

I 72 h

8 .0 

# I PUBLIC COPYStudy nnmbe CAS number : page 25 of 41 Teat Item ---- RESULTS Test itemconcentration [mg/L] : Control (test conditions as hr filtered teat item with itltration) eplicate Cell density [cella/mL]

(initial cell density = 5000 cells/mL) 24 h 48 h 72 h Growth (b) [~~1 of biomaee ] Growth ~~ (r) 1 23333 130000 540000 410833 1 .56 1[[ 26667 123333 593333 434167 1 .59 III 30000 150000 630000 482500 1 .61 IV 20000 126667 593333 430833 1 .59 V 30000 140000 673333 494167 1 .63 VI 30000 123333 586667 434167 1 .59 0 26667 132222 602778 447778 1 .60 Growth (b) Area under growth curve (0) : 447778 Percentage inhibition ofcell growth . 0 .0 Growth rate (r) Specific growth rate (0) . 1 .60 Percentage inhibition of cell growth rate . 0 .0 

## An pHvalues

## alysis 

# I Oh 8 .1 

# I I 72 h

8.4 

# I PUBLIC COPYStudy number : Teat Item CAS number page 26 of 41

RESULTS Test itemconcentraNon [mg/LI : 100 (8ltered, nominal) Cell density [ce1L/mL] Growth (b) (initial cell density =5000 cells/mL) [~~of Growth ~~ (r) Replicate 24 h 48 h 72 h biomaes 1 30000 110000 400000 327500 1 .46 11 30000 100000 380000 307500 1 .44 III 26667 130000 340000 314167 1 .41 0 28889 113333 373333 316611 1 .44 Growth (b) Area under growth curve (0) . 316611 Percentage inhibition ofcell growth . 29 .3 Growth rate (r) Specific growth rate (0) Percentage inhibition ofcell growth rate pHvalues Analysis 

# I Oh 8.2 1 .44

10.0 

# I I 72 h

73 

## 7 

# Ipage 27 of 41 Study number :- Teat item CAS nnm . 

# COP Y SUMMARY OF RESULTS Mum cell deaft duringthe tat Tat ihm coaceatrstloo [nWLl Cell deedty [=111/10L1 (initial cell dene#y = 5000cxlle/mL) 24Y 4

Sh 72 Control 32222 133000 588333 0 .9 27778 132222 604444 1 .9 30000 140000 358889 4 .3 36667 135556 444444 9.4 27778 147778 324444 20 .7 28889 80000 371111 45.3 23333 47778 113333 100 24444 42222 73333 Mom growth (b) [istegrd of blomm] Tat item coaeentradoe [MWL1 Area ander

. growth carve Effect [%) (Iaereare (-)) Control - 448889 0.0 0.9 449722 -0 .2 1 .9 336944 24.9 4 .3 381944 14 .9 9 .4 323278 27.5 20 .7 281944 37.2 43.3 113278 74 .3 100 90833 79 .8

Mean Qr+owlb rate (r) Tat ftem concentradon [N191L1 Growtr rate [1/d] Effect 1'K]

(Lcreme (_)) Control 1 .59 0 .0 0 .9 1 .60 -0 .6 1 .9 1 .42 10 .4 4 .3 1 .50 5 .9 9.4 1 .39 12 .3 20 .7 1 .44 9 .7 45.3 1 .04 34 .3 100 0 .90 43.7

/ 1 7Study number

Test Item CAS number : page 28 of 41



# PUBLIC

--- 

# COP Y SUMMARY OF RESULTS Mean cell denrity during the teat

Tat item concentratlon [~1 " Cel1 deadty [celWmL] (untial cell densaty = 5000cells/mL) 24h48

~ 72 Contool (test Conditions as for 997 26667 132222 602778 test item with filtration) 100 (filtered, nominal) 28889 113333 373778

Mean growth (b) [Integral of Nowngn) Test item

concentratlon [IMEN Area under growth cam Effect [%] (Inenue (-)) Control (tat condition. um

for 447778 0 .0 Slftred tat item

with filtration) 1 00 (MW4 nominal) 316611 29 .3

Mean growth rate (r) Tat item concentratlon 

### [MBIL] Growth rate



### [vd] EReet ['/o]



### (Increase ()) CoMol (test conditions : for 1 .60 0 .0 tilte+ed teat item

with filtration) 100 (filtered, nominal) 1 .44 10 .0p. 34

- . Study number : Test Item . CAS number : page 29 of 41

RESULTS Analysis of the growth and growth rate of the algal population within the 72 h exposure period (by probit analysis and Williams Multiple Sequential t-test procedure test's, respectively) give the following results: Results [mg//L] : 95% confidence limits [mg/L] EbC 50 (0-72 h) : 23 12-52 EbC 10 (0-72 h) :2.3 0 .14-5.6 F .,C 50(0-72 h) :134 72 - 872 E,C 10 (0-72 h) :8.9 0 .76-18 NOEC [b] :0.9 NOEC [r] :0.9 The test item

was insoluble in water. Treatment in an ultrasonic bath (5 min .) resulted in black colouration of the flask content, indicating fine dispersal of the test item . After treatment on a magnetic stirrer for 1 h, the suspension was tested without filtration. The results are expressed in terms of nominal concentrations. Microscopic investigation ofthe test suspension after 72 h revealed that algal cells were in free suspension. Very few were observed attached to flocs or lumps ofaggregated test item This indicated that "trapping" ofalgae in test itemdid not contribute significantly to growth inhibition. Ablank suspension ofthe test itemwas incubated as a blank value . When counting algae with the microcell counter, measurements were corrected for this blank value. In addition, a filtered solution of nominally 100 ere tested. Treatment was identical to direct addition, but after ultrasonificatio ' g in algal medium(24 h) the suspension was passed through a folded filter (7-12 itnmpore size) . The clear filtrate was then used in the algal test and resulted in 10.0% inhibition ofgrowth rate compared to control . 

# PUBLIC COPY /19Study number

Test Item CAS number : page 30of 41

RESULTS The concentration effect curves for DPsmodesmns subspicatus ate given in flgares 1 and 2, the growth curves are presented in figures 3 and 4 . The nutrient mediumfor pre-cultures and test cultures is given in annex 1 . The equations used to calculate the algal growth and growth rates as well as their percentage inhibition are presented in annex 2

. NOW

and LOEC determined according to Williams Multiple Sequential West procedure are presented in annex 3 . 

# PUBLIC COStudy numbe Test item page 31 of 41 CAS number : ARCHIVATION OF DOCUMENTS ANDTEST ARTICLES Study plans, raw

data and study reports are archived at the GLP archives ofthe test facility. Test articles are archived The sponsor must ensure that information submitted about the test item(identity testfng or information about physical and chemical properties) is complete and that it complies to the principles of Good Laboratory Practice. The cust~ +++» >a^ ~~~n a ~ le of the test itemused where sampling has not been performed 

# PUBLIC COPp. 37 Study number Test Item CAS number Figure 1 : page 32 of 41

Concestrado curve for Desnodesnus subspkaw exposed for 72 boon to iiim" ;Wed on growth (b) . Results of the probit analysis: Selected effective concentrations (ECx) ofthe test item and their 95%- confidence limits Parameter EC10 EC20 ECSO Value [mg/L] 2.279 5.061 23.087 lower 95°/

.-cl 0

.144 0

.787 11 .920 upper 950/6-cl 5.649 10.193 52.164

, "" 

# 0 To - go go-- 40 -- 30-- _ ZO 10-- / 0 . 0 .1 1 0 7 0 10 00 Conoontntlon [mg/L) na Iunalbn

__ 95%-CL 

# PUBLIC COPYStudy number Test Item CAS number : Flgare 2 : page 33 of 41

Concentrado curve for Desmodesmin subspkahn exposed for 72 hours to HN&ased on growth rate (r) . Results ofthe probit analysis: Selected effective concentrations (ECx) of the test itemand their 95°/ .- confidence limits Parameter EC10 EC20 EC50 Value [mg/L] 8

.859 22.590 134

.082 lower 95"/

.-cl 0

.763 6

.572 72.114 upper 95°/

.-cl 18.429 38

.730 872.235

100 i0 so To . - Go

# - so

# 40. ao :o to 1o 100 1000 101 00 1000 00 Concentration [mg/1.1 0 Date IYIIOMOq " G% -CL 

# PUBLIC COPYFigure 3 : Growth curves



### s

. ; 100 low,

24 48 n tcWAMI -O-a9mWl. --w-4a ny#L -rr-9 .4=Wt -F-zo

.7mWrt. -t4s.s m@Ot. i00mOt. 

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Study number Teat Item CAS number . Figure 4 : Growth carve: 

# 1 1000

100 10 1 0 hoan

-~Cootol (lae eaeomanlios nhr SYwedad tlom wYY1hmdm

--F100(mmfed Sllemd) page 35 of 41



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Study numbef

_ : Test Item CAS number N

. ANNEX 1 Nutrient medium

of pre-cultures and test cultures page 36 of 41

Concentration Final concentration in the Nutrient in stock solution solution of the precultures and test cultures Stock solution 1 : macro-nutrients NH4C1 1 .5 g/1 15 mg/1 MgCl2 x 6 HZO 1 .2 g/1 12 mg/1 CaC12x 2 Hz0 1 .8 g/1 18 mg/1 MgS04 x 7 H20 1 .5 g/1 15 mg/1 KH2P04 0 .169/1 1 .6 mg/1 Stock solution 2: Fe-EDTA FeC13 x 6 Hz0 80mg/1 80 pg/1 NazEDTAx 2 HZO 100 mg/1 100As/1 Stock solution 3: trace elements H31103 185 mg/1 185 pg/1 MnCIZ x 4 Hz0 415 mg/1 415 pg/1 ZnClz 3 mg/1 3 pg/1 CoC1s x 6H20 1 .5 mgll 1 .5 pg/1 CuClz x 2 HZO 0 .01 mg/1 0 .01 Itg/1 Na2Mo0,, x 2 Hz0 7 mg/1 7 pg/1 Solid NaHC03 is added to the nutrient media to make up a final concentration of 50 mg/L in the solutions ofthe pre-cultures and test cultures . Water hardness of the final nutrient medium is 1 .3 °dH, corresponding to 22 .5 mg/L CaCo3. 

# PUBLIC CO )tf / ;- 6p. 42

Study number Test item CAS nnmbe . ANNEXZ/1 The area under the growth carve (A) is calculated according to Equation [1] A= Ni ZNo Xt1+ Ni+ 2s-2No x(tx-t,)+ N2+ 23-2Ne x(b-tx) page 37 of 41

where tl is the time of the first measurement after the beginning of the test (24 h), t= is the time of the second measurement after the beginning of the test (48 b), t3 is the time of the last measurement at the end ofthe test (7Z 6) . No is the nominal initial cell density, Ni, N=, Ns is the measured cell density at time ti, t2, ts. Fromthe mean values of A for each test concetration the percentage inhibition is calculated according to Equation 121 I~,= A`-Aj x100

A4 where In, is the percentage inhibition (area) for test concentration i, A, is the mean area for test concentration i, A . is the mean area for the control .p. 43

Study number Test item CAS number

ANNEX 2/2 page 38 of 41

The growth rate [rr for each test concentration and for the control is calculated according to Equation (3] In Ns - In No r= b From the mean values of r the percentage inhibition of growth rate for each test concentration is calculated according to Equation 14]

1r i - ro-r+ x100 re where Irl is the percentage inhibition (growth rate) for test concentration i

is the mean growth rate for test concentration i r. is the mean growth rate for the control .p. 44 Study number Test item page 39 of 41 CAS number :

ANNEX3/1 Determination of NOECand LOEC based on growth (b) according to Williams Multiple Sequential ttest Procedure Comparison oftreatments with "Control" by the t test procedure after Williams. Significance was Alpha = 0 .05, one-sided smaller, Mean: arithmetic mean; n : sample size; s : standard deviation; LhM : max . likelihood mean; °/

.MDD : minimum

detectable difference to Control (in percent ofControl) ; t: sample t; t* : critical t for Ho: p1= IL2 =. . . = * the differences are significant in case (1 >jt*j (The residual variance ofan ANOVAwas applied; df = N- k ; N : sum of treatment replicates n(i) ; k : number of treatments) . Treatm . [mg/LJ Mean s df Lb1Vi " / .MDD t t* Sign. Control 44.889 6

.29290 0 .900 44.972 6

.29290 19 44.972 -17.139 0 .02 -1 .73 - 1 .900 33.694 6.29290 19 35 .944 -17.813 -2.01 -1 .80 + 4 .300 38 .194 6.29290 19 35.944 -18.041 -2.01 -1 .82 + 9 .400 32.528 6.29290 19 32.528 -18.121 -2.78 -1 .83 + 20 .700 28.194 6.29290 19 28.194 -18.200 -3 .75 -1 .84 + 45.500 11 .528 6

.29290 19 11 .528 -18.200 -7.50 -1 .84 + 100.000 9

.083 6 .29290 19 9

.083 -18.230 -8.05 -1 .84 + + : significant; -: non-significant A NOEC of 0.900 mg/L is suggested by the program . 

# PUBLIC COPYANNEX 3/2 Determination of NOEC and LOEC based on growth rate (r) according to William

Multiple Sequential t-test Procedure Comparison of treatments with "Control" by the t test procedure after Williams . Significance was Alpha =0 .05, one-sided smaller, Mean: arithmetic mean; n: sample size; s: standard deviation; LhM : max. likelihood mean; °/ .NmD : minimum

detectable difference to Control (in percent of Control) ; t: sample t; t'° : critical t for Ho : tt 1= p2 Itk ; the differences are significant in case 14 > It*I (The residual variance of an ANOVAwas applied; df = N- k; N : sum oftreatment replicates n(i) ; k: number of treatments) . Treatm . [mg/L] Mean s df LhM "hMDD t t* Sign. Control 1 .580 0

.09044 0 .900 1 .598 0

.09044 19 1 .598 -6 .999 0

.28 -1 .73 - 1 .900 1 .424 0

.09044 19 1 .459 -7.275 -1 .89 -1 .80 + 4 .300 1 .494 0

.09044 19 1 .459 -7.368 -1 .89 -1 .82 + 9 .400 1 .388 0

.09044 19 1 .409 -7.400 -2.67 -1 .83 + 20 .700 1 .430 0

.09044 19 1 .409 -7.433 -2.67 -1 .84 + 45.500 0

.997 0 .09044 19 0

.997 -7.433 -9.12 -1 .84 + 100.000 0

.891 0 .09044 19 0

.891 -7.445 -10.76 -1 .84 + + : significant; -: non-significant A NOECof0.900mg/L is suggested by the prograaLp. 46 page 41 of 41 PI 

# IQI 10 or)nxl

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w On~i~~~1~YA6sSTUDY FOR THE SIGN SENS1TiZATION EFFECT IN GUINEA PIGS (Guinea Pig Maximization Test according to Magnuason and Kligman) 

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Study Completion Date: March 30, 2007

Page 1 of 25GLP COMPLIANCE STATEMENT

Except for the following deviation, this study was conducted in compliance with the OECD Principles of Good Laboratory Practice as revised in 1997 (ENVIMCICHEM(98)17) and with the revised German Principles of Good Laboratory Practice according to Annex I German Chemicals Act (Bundesgesetzblatt Part 1, No. 40, issued June 27, 2002) . The deviation was as follows : Analytical determinations of On stability of the formulations in vaseline for - administration were not performed. . This deviation did not limit the assessment of the results. 

# PUBLIC COPYINDEX 1 . QUALITY ASSURANCE STATEMENT Z. SIGNATURES 3 . SUMMARY 

### 4 . INTRODUCTION 5. STUDY IDENTIFICATION AND RESPONSIBILITIES 5 .1 . Study Identification 5 .2. Responsibipdes 8. MATERIAL AND METHODS 8 .1 . Test item 5 .2. Test System 8.2 .1 . Speckmand Strain 8.2.2 . Accqrnattntlon 8 .2 .3 . State of Health 8 .2.4. Age and Body Weipht 8.3. Animal Care 8.3 .1 . Animal Ac©ommodalbn 6.3.2 . Animal Rooms 6.3.3 . Cleaning, Disinfect(on 8.3.4. Climatic Condftkm 8 .3.5. Diet 8 .4 . Test Methods 8.4 .1 . Guidelines 8.4 .2. Methodological Rellsbft 

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8 .5. Pwfbrmanoo of the Test 8 .5.1 . Test item Formulation 6.5.2. Grouping and Identillccatiion of Test Animals 8.5 .3. Dose Selecdon 8 .5.4. Sensitization Induction 8 .5.4.1 . First Induction ((ntradermat and topical) 8 .5 .4.2. Topical Induction 8.5.8 . Topical Challenge of the Sensit(zadon . 6.5.8 . Grading of the Skin Reaction 8 .5 .7. Evaluation of the Results 8 .5 .8. General Examinations ' 8 

### a 8 10 10 11 11 11 11 11 12 12 12 12 13 13 14 14 14 18 15 15 15 18 18 18 17 17 18 18

## ?.RESULTS 7.1 . General Examinations 7.2. Test for Skin-Sensitizing Effed 19 19 19 8. DISCUSSION ANDASSESSMENT Zp 9 . REFERENCES 

## 10 . ABBREVIATIONS 21 

## zz 11 . ANNEX 28 11 .1 . Individual Ran9e-flnding (PNot) Test Results 23 11 .2 . Skin Findings of Challenge 24 11 .3. Body Weights Zif 

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### Quality Assurance Statement Teat Item: Study No

. : Study-based inspections were conducted by the Quality Assurance on the dates given below . Audit reports have been submitted in writing to the study director and, if necessary, also to the laboratory management, or other persons affected. Date of Study phases Date of report to study director Inspection inspected mod/or management Jan. 25, 2007 STUDY PLAN , Jan, 23, 2007 Jan . 30, 2007 PREPARATION OF FORMULATIONIFEEDMIXTURE, Jan. 30, 2007

ADMINISTRATION, RAW DATA, MEASUREMENTS Mar . 29, 2007 - Mar. 29, 2007 FIRST DRAFT Mar. 29, 2007 Mar. 29, 2007 FINAL DRAFT Mar. 29, 2007 

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The results of the study and the methods used have been correctly reported.p. 52

2. SIGNATURES

Study Director

Head of Molecular and Special Toxicology: 

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3 . SUMNIARY The Guinea Pig Maximization Test (GPMT) was performed in 2007 on 30 female guinea pigs (20 ani Ift up and 10 control animals) to determine whether the test i ~~ ;ontent: 95.896) exhibits skin-sensitizing properties. The study was conducted according to OECD Guideline No . 406 ; EC Guideline S8/54/EC (22nd Adaptation of Guideline 871848/EEC) ; Health Effects Test Guideline, OPPTS 870.2800 with the following test itemconcentrations : First induction: 23.896 Second induction: 23.8% Challenge: 23.896 The test itemwas formulated in Vaseline to yield a paste. 

### The challenge with the 23.896 test item concentration led to no skin effects in the animals of the test itemgroup and the control group. In summary, under the conditions of the maximization test and with respect to the evaluation criteria the test itemtherefore exhibits no skin-sensitization potential .



# PUBLIC COPY4 . INTRODUCTION The test ite s tested for skin-sensitization properties on guinea pigs u st of MAGNUSSON and KLIGMAN 11, 21. 

### ain`d Kligman methodology applied at ave been verified [3], and are

Me are filed in the

the final report a re retained in the archives specified by

. sponsor. 

### he storage of a retention sample of the 'erence item is in the responsibility of the S . STUDY IDENTIFICATION AND E8PONSIBILITIES 5 .1 . Study Identification Study number : Experimental starting date: January 30, 2007 Experimental completion date: February 23, 2007 Study completion date: see signature pa8i Sponsor: 

# PUBLIC COPY5_ .2-ftaponsibiii ' Head of test facility: Head of Molecular and Genetic Toxicology: ' Study director : Head of Quality Assurance Unit: Archiving of study data:



# r

'JIUBLIC 

# CO8 . MATERIAL AND METH ODS 6.1 . Test Item Test item : Synonym : Batch No . : Chemical name: 10 p. 56

Content : 95 .8% Approval : until June 09, 2007 Physical state: solid Appearance: black powder Storage' : room temperature 'due to product Information given by the sponsor



# PUBLIC COPYp. 57

0 .2. Test System 8.2.1 . Species and Strain The animals used were SPF-bred guinea pigs of the strain Cri : HA supplied by the Charles River Laboratory Animal Breeders in 88353 MUM, Germany. A total of 30 female animals were used in the study. In addition, 5 female animals were used for the range-finding test for the induction concentration. = Appropriate sensitivity of guinea pig for sensitization tests and appropriate reliability of the experimental technique have been verified (3j and are - checked af- re - ular filed In the archives specified by 8.2.2. Acclimatization Following receipt,, the animals scheduled for this study were adapted to the maintenance conditions for a period of at least five days before treatment, and their state of health was monitored. 8=2.3 . State of Health Only healthy animals exhibiting no clinical signs were used for the study. The animals were not vaccinated or treated against Infections either before receipt, or during the adaptation or study periods. The females were nulliparous and non-pregnant . 6.2.4. Aae and Body laht The guinea pigs exhibited a weight range of 338 - 390 grams at the beginning of the study (see page 25) . The age of the animals was 5 - 8 weeks.



# PUBLIC COPYp. 58

6.3. Animal Care

' 63 . 1 . Animal Accommodati on During the adaptation and study period the animals were conventionally kept in Noryl cages [4) by Tecniplast Deutschland GmbH, 82382 Hohenpei(3enberg, Germany, in groups of eight during the adaptation period and in groups of two or five per cage throughout the study period. The cages were exchanged for ones with clean bedding at least two times per week. . Low-dust wood shavings named LITALABO supplied by SPPS, F 95100 Argenteuil, France were used as bedding. The wood shavings contaminant levels. Records of these test results are filed The analytical results provided no evidence for an effect on the study objective. 8.3.2. Animal Room

s All animal§ used in this study were kept in the same animal room, one In which guinea pigs fromother sensitization studies were also housed. Adequate separation, unambiguous cage markings and suitable organization of the work ensured that animals were not mixed up . 8.3.3. Cleanino. Disinfecti on The animal room

was swept out with a broom each day, and thoroughly cleaned with water once weekly. The room was disinfected at least once each month with Terralin® (working dilution 196) . Contamination of the diet and contact with the experimental animals were avoided during this work. No pest control measures were carried out in the animal rooms. KILLGERMRoach Traps®, a cockroach trap which uses no pesticides, was placed in the animal room . 

# PUBLIC COPY8.3.4. Climatic Conditions

The room

climate in the animal room was set as folkwn: Room temperature: 22 +l- 3°C (possibly drifting higher at outdoor temperatures above 24°C) Relative humidity: 40-70% Light/daq cycle: . Twelve hours rhythm ; artificial lighting Air exch~ge rate: >= 10 times per hour Occasional deviations from these standards occurred at times Including the period following the cleaning of the animal room . They had no apparent effect in the course of the study.

. . .DI The diet consisted of "PROVIMI KLIBA 3420 - Maintenance Diet for Guinea Pigs" supplied by PROVOMI KLI13A AG, and of tap water. Food and water were provided ad libitum . 

### The feed was maintenance diet for guinea pigs with the following content : Compound : Grain and grain-products High protein-containing vegetable products Milk products High energy products Raw fibre products Minerals, Amino acids, Vitamins Content : 19% Raw

protein 18.596 Raw fat 4.096 31% Convertible energy 1 96 rabbitlguinea pig 10.8 MJlkg 196 Phospor 0.5496 4496 Raw

ash 8

.996 Raw fibre 17.5% 496 Calcium 0.8396

Lysin 1 .0896 These are standard diets with selected nutrients and free of pathogens. The stainless steel dispensers for the pellets were cleaned at least once during the study and in case of visible contamination. Two or three 700-ml polycarbonate bottles per cage were used for water during the test period (4, 5] . The tap water met drinking water standards [8] . Records of the anahtses to monitnr com liance with th AA ki

` p e n ng water specfication are filed at he available data provided no evidence for an effect on the ottles were rinsed at least 2 times per week with tap water.

### B

.4. Test Methods 8.4.1 . Guideli nes OECD Guideline for Testing of Chemicals; Section 4: Health Effects, No. 406 : "Skin Sensitisation" [7] . EC Guideline 96/54/EC (22nd Adaptation of Guideline 671548IEEC) : Classification, Packaging and Labeling of Hazardous Materials, "Skin Sensitisation", Method B.B. 181 . Health Effects Test Guidelines, OPPTS 870.2600 Skin Sensitization: EPA 712-C-03- 197, March 2003 [9] . 8 .4.2. Methodological Rellabilltv The Guinea Pig Maximization Test methodology was checked for reliability in a test on female guinea pigs using alpha hexyl dnnamic aldehyde formulated in polyethylene glycol 400 at the concentrations indicated below . For the intradermal Induction a 596 test item formulation was used, for the topical induction a 2596 formulation and for the challenges a 12% and a 896 formulation . After the first challenge with a 1296 test itemformulation 10096 of the test animals and 80%of the control group animals exhibited dermal reactions in the challenge treatment . After the second challenge with a 896 test itemformulation 9096 of the test animals exhibited dermal reactions in the challenge treatment. There was no reddening of the skin to be observed on control group animals (3] . The sensitivity as well as the 'reliability of the experimental technique is thus confirmed by this study.. .~_ p. 61

6,5- Perfflrmance of the Test 8.5.1 . Test item Form

ulation 15 

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The test item was formulated in vaseline. For each animal 250mg test Item was mixed with 1 .0 ml (= 800 mg) vaseline. The formulations were visually described as paste. The vaseline was warmed at 37°C in hot water before use and was kept warm on a hot plate at approx. 40°C . Formulation of the test item In one of the usual vehicles was not possible. Therefore, the test item was formulated in vasiline and tested at the highest concentration possible. Hypoallergenic patches with the paste were also used for the first induction (see 6.5.4.1) because intradermai injection of the formulation was not possible. 6.5.2. Grouoina and Identification of Test -Animals Two groups of animals were allocated by random selection to the main study: one test itemgroup consisting of 20 experimental animals (nos. 11 - 30) and one control group made up of 10 animals (nos. 1 -10) . The randomization list was generated on a computer system by the program RandomV3.exe. The animals were Identified using cage cards specifying the test item, study number, animal number, dose and sex, and by individual markings using aqueous picric acid solution. 0 .5.3. Dose Selection The doses for the Induction and challenge treatments were selected on the basis of the results of the dose range-finding study presented In the Annex (see pages 23) . The following concentration was chosen for the main study: First induction: 23 .8%(= 250 mg test item/animal) Second induction: 23.896 {= 250 mg test itemlanimal) Challenge: 23.8%(= 250 mg test Iternlanimai)8.5.4. Sensitization Induction

.

8.5.4.1 . First Induction (i ntradermal and topical) 

# PUBLIC COPY The dorsal region and the flanks of the guinea pigs were shorn one day prior to the application. Starting behind the nape of the neck, two injections each in a rowwere made on the left and the right side of the spinal column. Hypoallergenic patches (2 cmx 4 cm) were placed between the injection sites, covered with aluminium foil and held securely in place for 24 hours on the skin using a ORABAND® selfadhesive tape (Fa. Orafoo . - The animals were treated as follows: Four injections (craniaUbilateral) : complete Freund's adjuvant (DIFCO LAB.) diluted 1 :1 with sterile physiological saline solution. The test item was not applicable to injections. The volume applied per injection site was 0 .1 ml . The patches were treated as folkma: ' a) Test Itemgroup: 250 m 06W0 ml vaseline b) Control group: 0.5 mi vaseline At the end of the 24-hour exposure period, the remaining test itemwas removed with sterile physiological saline. ' The Injection sites and the treatment area between the injections were visually assessed 2 and 7 days after the injection. 8.5.4.2. Topical Induction The topical induction was performed one week after the Intradennai induction. One day prior to topical treatment, the test areas of the animals were shorn . Hypoallergenic patches (2 cm x 4 cm) were placed between and on the injection sites, covered with aluminium foil and held securely in place on the skin using a ORABAND®self-adhesive tape (Fe. Orafol) . The patches were treated as toll a) Test itemgroup: 250 .0 ml vaseline b) Control group: 0.5 ml vaseline At the end of the 48-hour exposure period, the remaining test Itemwas removed with sterile physiological saline. 

## /`/7

### 8.5.5. Toaical Challenae of the Sensitization The challenge was performed three weeks after the Intradermal Induction. The dorsal region and the right flank of the animals were shorn one day prior to the challenge. During the challenge a hypoallergenic patch loaded with the 23.896 test item paste was placed on the right flank (caudal) of the animals of the test Item group and the control group and held securely in place on the skin with an ORABAND® self-adhesive tape for 24 hours. A patch only loaded with 0.5 ml vaseline was placed also on the right flank (cranial) as control . At the end of the exposure period, the remaining test itemwas removed with sterile physiological saline and twenty-one hours later the treated areas were shorn In the zone of the challenge area. 8.5.8. Gradino of the Skin Reaction The skin reactions were assessed 48 and 72 hours after the start of the application to induce the challenge and for the range-finding studies to establish concentrations for the topical induction and challenge in accordance with the following scoring system[1] : 

### 0 = No reaction 1 ~ Slight localized redness 2 ~ Moderate confluent redness 3 = Severe redness and swelling Descriptions of any dermal findings not covered by this awls were recorded.



# PUBLIC COPY6.5.7. Evaluation of the Resul ts

The number of, test item and control group animals exhibiting a reaction and the intensity of the reaction were determined and compared to assess whether sensitization occurred and if so, to what extent. The criterion for existence of sensitization was taken to be the occurrence of skin reactions In the test itemgroup animals at a higher incidence and greater intensity than in the control animals. A substance is interpreted to be sensitizing if by comparison with the control group 3096 or more of the test group animals reacted positive [8] . , 8.5.8. General Examinations The animals were observed for clinical signs at least once daily throughout the entire study period . The body weights of the animals were recorded by using a Mettler Toledo scale with printer LC-P45 on day 1 before the first induction and at the end of the study (see page 25) . 

# PUBLIC COPY7. RESULTS

7.1 . General Examinations Appearance and behaviour of the test itemgroup were not different fromthe control group. At the end of the study, the mean body weight of the treatment group animals was in the same range than that of the control group animals (see page 25) . After the intradermal induction of FCA (first induction) the animals in the control group and test itemgroup showed after 48 hours at the injection sites: red wheals After 7 days the following effects were recorded at the injection sites in the control group and in the test itemgroup: wheals and encrustation. There were no skin effects (redness) on the treatment areas between the injection sites (FCA) of the first induction in the control or test itemgroup after 48 hours and 7 days. 7.2. Test for Skin-Sensitizina Effect The incidence of skin reactions following the challenge is summarized in the following table: Table: Number of animals exhibiting skin effects

Test itemgroup (20 animals) Control group (10 animal,) Test item patch Control patch Test item patch Control patch Hours 48 72 Total 48 72 48 72 Total 48 72 Challenge 23.896 0 0 0 0 0 0 0 0 0 0 

### For individual animal challenge exposure results see page 24 in the Annex . 

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# 0"0 20

. 400"-N"~) 8. DISCUSSION ANDASSESSMENT In a Guinea Pig Maximization T examined in female guinea pigs for its skin sensitizing rope . rs and second induction were performed using a 23.896 test itemconcentration . The test itemwas formulated in vaseline to yield a paste. After the first induction the animals in the control group and the test item group showed strong effects up to encrustation at the injection sites of the first induction . The challenge with the 23.896 test item concentration led to no skin effects in the animals of the test item group and to no skin effects in the animals of the control group. . In summary, by comparing the results in the treatment group and in the control group under the conditions of the maximizatiori test [7] and with respect to the evaluation criteria (8] the test item therefore exhibits no skin-sensitization potential . 

# PUBLIC COPY9. REFEREN

1 . MAGNUSSON, B

., A.M . KLIGMAN

: The identification of contact allergens by animal assay . The guinea pig maximization test . J . irnrost. Dermatol . 52, 268-278 (1989) 2 . MAGNUSSON, B

. : . Identification of contact sensitizers by animal . assay. Contact Dermatitis 8, 46-50 (1980)

4 . MEISTER, G

: Versuchstierk8fige for die Haltung und Zucht Yon Kleinnagem . Zschr . Versuchstierkunds 7, 144-153 (1965) 5 . SPIEGEL, A

., R . GONNERT: Neus KBfige for MBuse und Ratten . Zachr . Versuchsderkunde 1, 38-48 (1981) 8 . Trinkwasser-Verordnung vom 21 .05.2001 Bundesgesetzblatt, I Nr. 24 vom 28 .5.20018 .959 7. OECD-Guideline for Testing of Chemicals; Section 4 : Health Effects, No. 408

: "Skin Sensitisstion", adopted: 17th July 1992 8 . Commission Directive 98154/EC of 30 July 1998 adapting to technical progress for the twenty-second time Council Directive 87/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances; published in the Official Journal of the European Communities L 248Vol . 39, 30 September 1998 9 . U .S . Environmental Protection Agency (US-EPA), Health Effects Test Guidelines: OPPTS 870 .2800 Skin Sensitization EPA 712-C-03-197, March 2003 

# PUBLIC COPY10 . A88REVIATION~ approx. approximately confer centimetre e.g. for example Fa firm FCA complete Freund's adjuvant g gram ' h hour international units mean mg milligram ml millilitre mm millimetre No number nos numbers g standard deviation



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### 11 . ANNEX

11 .1 . Individual Range-finding (Pilot) Test Results Dose-r anoe finding study for inductions snd challenge In a dose-range finding study the 23.8% paste and the vehicle were tested in each case on five guinea pigs. The patches moistened with paste of the test itemor 0.5 ml vaseiine were applied to each animal under occlusive conditions for 24 hours. At the end of the exposure period, the remaining test itemor the vehicle were removed with physiological saline and twenty-one hours later the treated areas were shom . The skin reactions were evaluated 48 hours and 72 hours after the start of the application . The results are presented in the tables below(assessment criteria see 8 .5 .6) : Animal No. 0% 23.8%

48h 72h 48h 72h 1 0 0 0 0 

### 2 0 0 0 0 3 0 0 0 0 

### 5 0 0 0 0 5 0 0 0 0

Selection of dose: Based on the results of the dose range-finding study, the following concentration was selected for the induc8ons and the challenge: First induction: 23.896 Second induction: 23.8% Challenge: 23.896

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### 24

11 

### . 2

### . Skin Eindinos of ChalleM2 

# PUBLIC COPY Challenge with a concentradon of 23 .8°ib : Test itempatch Control patch Animal 48h# 72h# 48h# 72h# No, Control grou p 

### 1 0 0 a 0 2 0 0 0 0 3 0 0 0 0 4 0 0 0 0



### 0 0 0 0 0 0 0 0 7 0 . 0 0 0 8 0 0 0 0 9 0 0 0 0 10 0 0 0 0 Test hem

grou p 11 12 0

, 0 0

0 0

0 . 0

0 13 0 0 0 0 14 0 0 0 0 15 0 0 0 0 18 0 0 0 0 17 0 0 0 0 18 ' 0 0 0 0 19 0 0 0 0 20 ' 0 0 0 0 21 0 0 0 0 22 0 0 0 0 23 0 0 0 0 24 0 0 0 0 25 0 0 0 0 28 0 0 0 0 27 0 0 0 0 28 0 0 0 0 29 0 0 0 . 0 30 0 0 0~ 0 W Findings made 48 and 72 h after start of exposure (assessment criteria see 8 .5.8)

### 11 .3. Body Webhts we hts Control rou edlndi group Test Um g roup Animal No

. 1 ` 25 Animsl No . 1 " 25 1 352 504 11 382 510 2 357 508 12 383 472 3 381 473 13 349 495 4 349 467 14 359 497 5 365 479 15 353 513 8 370 524 18 388 504 7 390 ' 542 17 379 467 8 338 473 18 359 510 9 372 498 19 348 482 10 384 531 20 382 522 M 384 500 21 374 518 3 15.9 26.8 22 347 457 23 382 528 24 354 520 25 371 490 28 355 500 27 353 473 28 353 519 29 346 504 30 359 523 M 382 500 8 12.5 20 .6 " at the day of the 1 st application

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Study CamPietton Date: Decemba 4, 2006

Page 1 of 27

### GLP Compliance Statement This stndy was conducted in compliance with the OECDPrinciples ofGood Laboratory Practice as revised in 1997 [ENV/MGCHEM(98)17] and with the revised German Principles ofGood Laboratory Practice according to Annex 1 German Chemicals Act (Buadesgesetzblatt Part 1, No 40, issued June 27, 2002) . 

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### Table of Contents l . Signatures.- . . .N

.tN .NNNNN .N .N .NNNNN .N . NNNN.NNNNNNNN .NNNNNS 2 . Swnmary .NNNNNNNNNNNN .N .NN NNNN.N

. .NNN. . . .N . .NNNNNN .NNNNN . . . . . . . . . .N .6 3" ID . . __"

_ " . . .QNNN

" N" N

" . .NNNN

" .NNN" N. . """ MNN.N .NN. " N

" N .NNNN NN . " N" .N

" N " NNN

" Y 4 . IdentlAatioa and R~/pOn/i~///1~~" NN. " NNMNNNNNN " . . " NNNN" N """" NNN

.NN " 9 4.1 Identification ofthe Study . . . . .. . . . . . .. . . .. . . .. . .. . . .. . . . . . . . . . . . . . . .. . . . . . . .. . . . . . .. . . . .. . .. . . .. . .. . . . . . . . . . . . .. . .. . . .. . 9 4 .2 Responsibilities. . .. . . .. . . .. . . .. . . . . . .. . . . . . . . . . . . . .. . .. . . .. . . . . . . .. . . .. . . .. . . . . . . . . . . .. . . . . . . .. . .. . . .. . . .. . . . . . .. . . . . . . . . . . .. . .9 S " 1 "

ja{erW

and MetboYs" NN "" NNNN" NN " . " N . .N .NNNN

" NN . " NNNNNN.N" NNN " N .N " NNNNNN

"" NNN

" N . " lU 5.1 Test Compound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10 5.2 Exposure Procedure .. . . .. . . . . . . .. . .. . . . . . .. . .. . . .. . .. . . .. . . . . . . . . . . . . . . .. . . .. . . .. . . .. . .. . . . . . . . . . . .. . . .. . . . . . . .. . . .. . . . . . .11 5.3 Application Dose and Exposue+e Period. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l l 5 .4 Teat System(Animals) . .. . . .. . . .. . .. . . .. . . . . . . . . . . . . .. . .. . . . . . . . . . . .. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . .. . . .. . . . . . . . .12 5.5 Housing Conditions . . . . . . . . . . . . . .. . . . . . .. . .. . . .. . . . . . . . . . . . . . .. . .. . . . . . . . . . . .. . . . .. . .. . . . . . . .. . .. . . . . . . .. . . . . . . . . . . . . . . .. .13 5.6 Test Methods . . . .. . . . . . .. . . . . . . .. . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . .. . . . .. . .. . . . .. . . . . . .. . . .. . . . . . . .. . .. . . . . . . . . . . . . . .14 

## 5.7 observations . . .. . . .. . . . .. . .. . . .. . . .. . .. . . . .. . .. .. . . .. . . .. . . .. . .. . . . .. . . .. . . .. . . .. . .. . . .. . . .. . . . .. .. . . . .. . . .. . . .. . . .. . .. . . .. . . .15 5.8 Data Collection. . . . . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . .. . . . .. . . . . . . . . . . .. . .. . . . .. . .. . . . . . . .. . . .. . .. . . .. . . . . . . .. . . . . . . . .16 5.9 Calculation ofthe LD50. . .. . . . . . .. . . . . . .. . . .. . .. . . .. . .. . . . .. . . . . . . .. . .. . . .. . . .. . . . . . . . . . . .. . . .. . . . . . . .. . .. . . . . . . . . . . . . .16

. . . Results N " N . .N "" NNN

.N " NN """ NNNNN.NN "" NNNN

.NN " N " N.N "" N

. . " . .NN

.N .N 17 6 .1 Dose-Response Table (LD50) . . .. . .. . . . . . . . . . .. . .. . . . . . . .. . . . . . . .. . . .. . . .. . . .. . . .. . . . . . . . . . .. . .. . . . . .. . .. . . .. . . .. . . .17 6 .2 Clinical Sips. . . .. . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . .. . .. . . .. . . .. . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . .. . . . . . . .. . . .. . . . . . . . .17 6.3 Body weights . . . . . . . .. .. . . .. : . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . . .. . .. . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . .. . . . . . . .. .17 6 .4 Gross PathologyFindinga. . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . .. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . .18 I " CoadrRYY " N " NNNNNNN" . .NNNN " N .NNN

"" NN

" N .NNN" NN

.NNNNNNNN

. .NN. .NNN

.NNN

"" NN

"" N " N "" N " N lI 8 . Appendis " N " N""" NN

"" N " N . .NN

. " N . " . "" NNN.NN .NN .NNNNN

.N " NNNNN

.N .N "" NNNNNNNN " N"" NN

" NNN

"" N_O List ofAbbreviations Used in the Appetft . . . . .. . .. . . . .. . . . . . . .. . . .. . .. . . .. . . .. . . . . . . . . . . .. . . . . . . .. . . .. . . . . . .. . . . . 20 Clinical Signs, Oroups . . . .. . . . .. . . . . . . .. . .. . . .. . . . . . .. . . . . . . . . .. . . . .. . . . . . . . . . . . . . . . . . .. . . .. . . .. . . .. . . . . . .. . . . .. . . . . . . . . .. . . . . . . 21 Clinical Sips, Individwal . . .. . . . . . . . . . . . . . . .. . .. . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . . . . . . .. . . . . . . .. . .. . . . . . .22 Body Weights . . . . . . . .. . . .. . . .. . .. . . . . . . . . . . . .. . . .. . .. . . . . . . . . . . . . . .. .. . . . .. . . .. . . .. . . .. . .. . . . . . . . . . . . .. . .. . . .. . . .. . . . . . . . . . . .. . . . . . . .23 Weight C3ain. . . . . . .. . . . .. . .. . . .. . . . . . . . . . . . .. . .. . . .. . .. . . . . . . . . . . .. . . . . . . . . .. . . . . .. . . . . . .. . .. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .24 Microscopic Findings. . .. . . . . . . .. . . .. . . .. . . . . . .. . . .. . .. . . . . . . .. . .. . . .. . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . .. . . .. . . . . . . . . . . 25 Statement of C1LP Compliance . . . .. . . . . . . . . . . . . . . . . .. . . .. . . . . . . .. . . . . . .. . . .. . . . . . . . . . .. . . . .. . . . . . . .. . . . . . . .. . .. . . . . . . . . . . .. 26 

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### OaaRtv Anumnee Statement Test Itew

: Study No . : In the relevant laboratory areas the equipment and atandacd processee used in connection with toxicological short-termstudies wmv audited periodically by the Quality Assurance (pmcesf- band inspecdona~ Audit reports ha" been submitted in wnW* to the study director and to the laboratory management. Date of Study plmsof Dote of report to study dimter Irupadoa 1a~pectod sadlor msuaRe®eat 

### Sept a, 2006 STUDY PLAN Nov . 29,20W CLINICAL OHBERYATION, Iv1BA3UR131MNf8 Nov. 29,2W6

(PROCESS HA9BD) Nov. 21, 2006 - Nov. 21, 2006 FIRST DRAFT . Nov. 21, 2006 Doo . 04, 2006 FINAL DRAFT Dec. 04, 2006 

# PUBLIC COPY (~M Dste: no results offt study am the methods used have how correctly reported.

### 1 . Signatures



## Study nkedor . 

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### 2 .

Tlre results an summarized in the table below . Table Z-1 Dow-R»ponw dow mplkg OOOdoolopikal ocarnmnoe of si~a fims of deeth moRwqt bw . resur t%l male 

### 2000 0 I 0 I a - - 0 female 

## zooo o I 0 I a - - 0 " number of anknNc which died sponhneowly endlor was aailla~d in mWibund stMo I number ofanknalt wilh WW* of tooddly I Ift number of W*rAft LOW Par Band an the present inv~ to be regarded to have tha following LD50values : - opp "p LD50 rat, male : > 2000mg/kg body weiQht 

### rat, hnale : > 2000m&ft body weight 3o it is regarded ac nontoxic aft dermal applicadon. (OH3 Category Sheerlassifled analogous OECDdraft guideline 434) 

# PUBLIC COPY'/ Groups of s male and s female wislar rob received s single dermal dose of 

## 2000 mift bodyweqft oftotest compound applied amocclusively for 24 lwum A dose of 2000m8/lcg body weight was tolerated by male sad female rats without mortalities, clinical sIgwtoxivologtcal effecte an vraig&developmat and Stow pathological 8udin8e. 1U method used complied withthe OECD-(inidoline for Taft ofChemicals No . 402, "Acute Dermal Toxicity", adopted:- 24 Febnury,1987; EEC Dirocdve 67/348 Annex V, Method 13.3. in its anz+eat v"on, and theprinciples of EPA Heft . Effects Ted Guidelines (OPPTS 870 12001 United States Enviromo~qd Protection Agency, EPA 712-C

" 98-192, August 1998) . 

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# r

Study initiation date : 2006-08-25 (YYYY-MM-DD) Experimental starting date : 200649-06 (YYYY-MM-DD) Experimental completion date : 20064)9-20 (YYYY-MM-DD) Study completion date : see signature pap The study objective was to determine scute toxicity after dermal application. The methodology ofthe study was based on the 0ECD41uideline for Testing of Cbemicals; Section 4

: Health Effects No

. 402, "Acute Dermal Toxicity", adopted: 24 February, 1987; Annex V, Part H .3. (acute toxicity (dermal]) to DirecNve 67/548/EEC ofthe Council ofthe European Communities of 27Jian% 1967 in its current version, and the principles of EPA Health Effects Teat Guidelines (OPPTS 870 .1200), Acute Dermal Toxicity (united States Enviromnentitl Protection Agency, EPA 712-C-98-192, August 1998) . The raw

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

storage ofa retention sample is in the responsibility o aponsor. 

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### 4

. 9 

### Identitieation and Responsibilities 4.1 Identification ofthe Study Study No. 

### 4 .2 ResponsibiUtier Head of Department Head ofTest Facility Arcbving No Heed ofQuality Assurance U ]jj Study Director 

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# s. 19



# Material and MeMods The "Acute Dermal Toxicity" descn'bod in OECDguideline 402 was used for judgment on the acute toxicity ofthe test substimce. For classification according to the Globally Harmonized Systemof Classification and Labeling ofChemical` ((1H3) do OECDdraft guideline 434was used. 5 .1 Test Compound Test compound Chemical name

: 9S . used for calculations) Expiry date : 2006-12-09 (YYYY-MM-DD) Conflrmadon ofthe identity ofthe tat compound was performed. Hatch no. Content oftest aubatanov Appearanoe : black, povvdery to coarse-grained Storage : room tomperattra 

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&2 Exposure Prmedurs One day before the stat ofthe trmftw the back aod flub ofthe rats were shoam (~proximately 10%

ofthe body surfal<x arm) . For each dose and animd the required amount of pure solid teat subamnoe was weigbed and transferred to a wd game-layer (6 .0 cm x 5.0 cm = 30 .0 cm') ofa Cutiplasto sberil" coated with ai~tight L,eukoflexo"` . The gmr~e Strip was placed on the rat's back and secured in place using Pehao-HaW cohesive stretch tape (8cm x 23 cm) and additionally covered with a "Lomir biomedical Ino ratjackct", which was connected witha safety pin to the straoch tape to ensure dud the animals could not ingest the test subatance. After 24 bows do dressings wen ramoved aod the am was rinsed with tepid water using soap and Sandy pdft the am dry. As the test substance was applied puro, the andytical examindion ofstability was not performed . 5 .3 Application Does md Exposure Period Depending on the body weight ofthe aaimals and the surface area on which the tag substence was applied, the following dose ranp (mg/cm) was applied (exposuc+e lasted for 24 hours) :

Table d-1 Dow

Rnqa dose (maAca b

.wJ surface Was mm of doses (makrrA male 

### 2000 20

.0om' 22

.1 - 24

.e 2000 20.o cm' 20 .5 - 22

.0p. 83



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5 .4 Test Systeas (Animsls) ne study was performed in Wiatar rats. The strain used was HadCpb:Wu (breeder . l ;arl8n/W

+*+kll .r. .nn G

.nbAl,' ~ 3 31,-7g Boxhen, Germany) . Animals ofthis strain have been toxicological studies for many years. Historical data on their phyalology and spontaneoas alterations is available. The state of health ofthe brooding colony is routinely spot-c>mclced for the main specific pathogens. The results ofthen examinations are archived At start ofthe study the animals were nulliparous and non-pregnant and free ofall clinical symptoms or diseases. T1re seelimatisatlon time in the animal room was at least S days. Body weights at start ofstudy: 221g - 246g 205 g - 220 g This is according to an age of 9-13 weelcs approximately. The animals were assigned to their groups by random~tion. T1w randomlist was based on evenly distributed chsnce numbers especially generated for the study by a software application. The animals were identified by labels on the cages stating study number, test compound, animal number, group number, etc. and by individual anima1 identification using permanent eldn marking.p. 84



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5 .5 Housing Condidou The animal moan had a standardized climate: 

## Room tenperstime : 22}20c Air humidity : SS fS'XX VentillItion 10changes per hour Lightl Dark cycle : 12 hours rhythm Occasional deviations from these standards oceurred, e.g. during cleaning ofthe animal room

. They did not have any apparent influence on the outcome ofthe study. The animal room

was sounded with radio program . The animals were caged individually in polycarbonate cages on low dust wood granulate bedding (J. Retaenmaier &s8hne, 73494 Rpsenberg, Germany) . The caps ofthe animals were placed on racks. The wood granulate wu randomly checked for contaminants at regular intervals and the results ha ve been stored at the analyses yielded no evidence ofany adverse effects on the aimofthe study. The cages were changed at lad once a week. Feed racks and water bottles were not changed. All cage material was washed with hot water. In the JIM

stage ofthe washing programs an alkaline cleaning agent (Neodischer Alka 300

; Chemi'che Fabrik Dr. Weigert (3mbH &Co . KO, concentration : 2

.2 g/1) was used. The animals received the standard diet "Provimi Kliba 3883.0.15 Maus/Ratte Haltnng, Kaiaeraugat Switzerland", and tap water ad libitumfrom polycarbonate bottles.p. 85

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The nutritive composition and the contaminent content ofthe standard diet were chccked and analyzed routinely in randomsamples. Nothing untoward was found. The tap water was ofdrinldng watet quality (according to the Drinking Water Decree in the cun+ent version) . The results ofthe analyses have been sto avsilable dats yielded no evidence ofany sim ofthe study. The food was available from racks in the lid ofthe cage, polycarbonate botNes w= used for drinking water. The animal room

was cleaned once a week and disinfected at least owe a month. A continuous pest control was pecformed using a cockroach trap without pesticides (e.g. KillgecmRoach Trap, KillgermC3mbH, 41460Neuss, Germany) . The contact ofthe animals with the traps was avoided in any case. 5.6 Test Methods The study was performed in accordance with the following guidelines: OECD - Guideline for Tesdng ofChemicals; Section 4 : Health Effects, No. 402 - "Acute Dermal Toxicity" ; adopted: 24February, 1987 ('Ilurd Addendum to the 1981 OECDGuidelines for Testing ofChemicals, OECD Publication Service, Paris 1987) ; Annex V, Part H.3

. (acute toxicity [dermal]) to Directive 67/548/EEC ofthe Council ofthe European Communities of 27 Am, 1967 in its current version, and the principles of EPA Health Effects Ted Guidelines (OPPTS 870 .1200), Acute Dermal Toxicity (United States Enviroame~al Protection Agency, EPA 712-C-98- 192, August 1998) .

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5 .7 Observations clinical signs and mortality rates were determined several times on the day of application and subsequently at least once daily for an observation period ofat least 14 days. Mortality and in the event of aymptoms owning, nature, duration and intensity were recorded individually. The day of application is defined as day 1 . T'unes after application undl the following daywere recorded either in minutes or in hours, depending on what was appropriate . Minutes were rounded in five-minutesteps according international agreements, e .g. 0' - < 2

.5' to 0', > 2.5'- < 7.5' to 5' and so on. Hours were rounded to full hours. In contrast to this, afurther observation intervals are given in days. Tbe duration of the symptoms and the times ofdeath are given relative to the time ofapplication to the individual animal . The real time points can be taken from the raw data. In general, death will be taken as a symptom . Due to die computer system used, death is not shown as a clinical symptom in the Ho of the appendix. If no symptoms are seen until death, time ofdeath will be talsen as the first occurrence ofa symptom . Inthe results section, the findings an summarized without any indication of intensity. The findings can be found for the groups and individual animals in the appendix. The weight gain ofthe animals was checked weekly until the and ofthe study. The weights are given in the tables in the appendix as individual and mean values. The means and standard deviations were calculated on the base ofthe rounded individual values . The weights are given in grams (g) . Indicated under the heading day 8 are e.g. the data obtained on the 7th day after application. Animals which died or were killed in moribund state were weighed (except on day of application) and dissected aa soon as possible, and examined macroscopically. The surviving animals were sacrificed by carbon dioxide at the end ofthe study, dissected and examined macroscopically. .p. 87

16 

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s.s- nsa couft-aoa During this shady for coUoctiozr, storage and evaluation of data a validated LAN-linlced compoter system was used, which is deaigaed and created in-house. If necessary to data w= collected oi&ne. Hardarare and operating systems: " HP-1000A (4x, operating systemRTE-A 6 .2) " HP-3000 series 900 (lx, operating system heErlX 5.5) " HP-9000 series 200 (lx, operating systemHP-UX 11 .0) Software : Data was stored on HP-3000in an HP'IurboImmagdXi . Database. 

### 5 .9 Calculation of the LDSO An assessment ofthe LDSOwas made based on the appUed dose and doee-response ctuve, respectively.p. 88

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6 . Reaulb 6 .1 DowRespoaseTable (LM) The reaulta ofthe study for acute damal toxicity in the rat, including the LD50, are summarized in the table below . Tabb i-1 Doae-Rraponaa d mpAcp bo ~

I occurrence d ~ipna *no Of death -

_ 1 male 2000 0 I 0 I S - _ 0 female 2000 0 I 0 I S - _ 0 ' numbK

of anknab which dad apondnaouay andlar ww~a sacrillosd in moribund sfaft I number ofanimala with signs of baoddty I bohl number ofanimals wad per group LDS0 male : > 2000 mqft bw . female : > 2000 mg/!cg bw . (aHS cateM

5/unclassified ralopom

OECDdraft guideline 4

34) 

### 6 .2 Ciinical Sips No clinical signs wffe oburved (see POWs on pap 21, individual aaimsls on page 22) . 

### 6 .3 Body Weiotr There wem

no toxicological effecta on body vidght: or body weight development in males and females

. Tbe slight daM=se in body Weightgain ofthree females in the Srst week is assumed to be caused by the atrees ofthe epplication procedut+e, and is not considered teat compound related. 'The weights am given in the appendix as individual and mean values on pap 23, for body weight gain 3ee page 24 .

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6 .4 Grow

Pst6oloiy Flndinp The neaopsiea performed at the end ofthe study revealed no particular findings (see aPPouft . Pap 25) .19 

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7 . Conclasioe the followinQLD50values: Hased onthe present iovestimptl be regarded to have LDSO rat, male . > 2000mg/lcQbody weight

rat, fanale : > 2000mg/kg body weight 3o it is regarded as nontoxic aRet decmal application. (GH3 Category S/unclassi8ed melogoos OECDdnft guideline 434)p. 91



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S . Appendix List ofAbbreviations Used In the Appendix In addition to the abbreviations for the basic units ofthe International Unit System (SI) stipulated by law, and designations for decimal multiples and parts of units, the following abbreviations an used: am . (aft meridiem) = before noon approx. appsoximabely bw . body weight C .A . Chemical Abstracts CAS Chemical Abstracts Service d day E final necropsy / Endsektion e.g. eacanpli gmtia (= for example) ff following h how LDSO median ledW dose m mean M moribund sacrifice max. intens. maximum inteneity n.a.o . no abnormality observed no. number pGrL partly p.m (pat neridiem) - after noon ct/CT dermal / cutan SD/a standard deviation T death / Tod time of d . time ofdeath ta treatment ares ' minute °A percent fr .cnale a malep. 92

clinical signs; groups 

# 21 Akut/acuta clinical signs I incidence ( duration of ( MX. ( ti~s of siona lntsns . death

. . . . . . .------ . . . . . . ._._. . . . . . . . . . . .----- . . . . . . . . . . . . . . .--------------------- " ----- 2000 q0/KO mala CT

---- . . . . . . .- " --- . . . . . . . . . . .--- " -------------- " ---- . . . .--- . . . . . . . . . . . .------------- n.a .o.

---- . . .------- " - " ------------ ""

---------- . . . ._ . .----- " --- . . . . . . . . . .--------- " --- . . 2000 NQ/KG feasts CT

-------------------- " ----- . . .------ . . . . . . . . .------------------ . . . . . . . . .- " -- . . . . . . . n .a .o .

# 22 clinical signs; Individual . Akut/scuto snissl I clinicst signs duration of I ~at . I time of no . I stem Intsns . death

----- . . . .------------ . . . . . .------------------------ " --- " -------------- . . . . . . . . . . . .

2000 MO/K" msls CT

-------- . . . . . . . . . . .------- . . . . .---- . . . . . . . . . . . .-------- . . . . .----------------------

2000 MO/KO fssslo CT

-- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .----------------------------- . . .--- . . .--- 6 n.s .o . 7 n.s .o . " n.s .o. 9 n.s .o. 10 n.s .o.p. 94 

# 23 TiarOwichta / bodir wi9hta (0) Akut/acuta

2000 MO/KO uinnlich / aala CT 1 : 3 " S 

# .

# i 1 238 2" 306 238 270 312 240 263 296 2" 266 297 221 249 297 237262 - 294 9 .3 8.0 21 .5 2000 MO/KO waibtieh / faoslo CT 6 213 217 229 7 212 208 227 8 20s 210 221 9 220 218 229 10 206 204 210

. 211 -~23

. 6.1 6.0 8.1 nae6 To~ T~a~ft after death time of d.p. 95 

# 24 Owiehtsentui¢klund / w11ht dn1n (d)

1 2 3 4 

### S



# . 2000 uo/Ka winnlieh / 

# i rle CT +bd 238 +26 +42 238 +32 +42 +74 240 +23 +33 +56 +S1 246 +20 +31 221 +28 +d +36 237 26 31 9 .3 4 .6 13 .9 _ _ 14 .9 2000 MO/KO wsibliah / f~le CT 6 213 +4 +12 +16 7 212 -4 +19 +1S +16 8 205 +9 +11 9 220 -2 +11 +9 10 206 -2 +6 +4 2 1 0 12 12 s 6 .1 4.0 4.7 5 " 3 

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# 25 1ndividual nacroscopic findinOs Alt findings

. .---- . . . . . . . .---------- " ------ " ------- . . .----- . . . .---------------------------------------- anisrl tisn / type finding no. of death

. . . . . . . . . ,- . .--t---------- " --------------------------------------- . . . . . . . .---- . . . . .--- 1 I 15d I II General observations I I no pathoLogicat finding I 1 2 1 1Sd / E I Osnaral observations I I no pathological find ins 7 1 19d / E I General observations 1 I no pathological find no 4 1 1Sd / E I General observations I I no pathological finding S 1 1Sd / E I General observations _-- .L_ I no pathological finding Sd / E onsra obsarvations

I no pathological finding 7 1 1!d / E 1 central observations I I no pathological finding 8 I 1Sd / E I General observations I I no pathological, finding 9 1 tsd / 0 I General observations I I no pathological finding 

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10 1 iSd / E I General observations 1 I no pathological finding

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Study Compledon Deoe: Decaaber 4, 2006

Pap 1 of 28Except for the following deviation this study was conducted in compliance with the OECD Principles of Good Laboratory Practice aa revised in 1997 [ENY/MGCHEM(98J7] and with the Ocumm Principles of Good Laboratory Practice according to Annex I Qarmen Cltiemicals Act (BUndesgesetzblatt Part I, No 40, issued June27, 2002) . The deviation was as follows: Stability ofthe test substance in the foirnulation for administration was not determined. However, the teat substance was formulated directly before administration. The tog substance char~cteristics we+e not suggestive of rapid reaction with the formulation agent.

. The deviation does not limit the aaxsmnent of reaulk 

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