p. 1 

Page 13 of 17 

Study number 

Test Item 

CAS number : 

RESULTS 

Test Item concentration [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 

pH value 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 COPY 
p. 2 

Page 14 of 17 

Study number 

Test Item 

CAS number : 

Teat item concentration [mg/L] : 

RESULTS 

100 

Abiotic parameters 0 h 24 h 48 h 72 h % h 

Temperature °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 COPY 
Study namber 

Test item 

CAS number : 

RESULTS 

Analysis of the 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 item was 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 

aquarium content, indicating very fine dispersal of the test item In the first hours of the test, 

fine flocs of test item formed and partly settled on the bottom of the test vessel. 

The results are expressed in terms of nominal concentrations. 

PUBLIC COPY 
p. 4 

Page 16 of 17 

Study number 

Test Item I 

CAS number : 

ARCHIVATION OF DOCUMENTS AND TEST ARTICLES 

Study plans, raw data and study reports are archived at the GLP archives 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 item used where sampling has not been 

performed 

PUBLIC COPY 
p. 5 

Page 17 of 17 

Study number 

Test item 

CAS number 4:. 

1 0 

_ Gpy 

GLP CERTIFICATE 

"L!/wNb~"Y~ b "V 4"mo%M* 

("" wmNh4q Y 6 HY MW I Obw"~YN- 

~W~Y+~aff AwmwhAAY"~~wr ~rrirI~~~OLtwi~~ 

~~yp~rY~rtl~clYrli CwrYirFrr~rICwrw~n7N "Cs 

o 

pa~ 1~bK~Ipal- Mwd6prMr 

~IIYd~AM~lM ~741'OlYw~l Ir~" W~4NY.fiO[m~~l 

"wool O / 1 

1fwOrIN 6y4 

d~Yr~AwFif~~air~irr"~r ~Ywr~~wtr~ru~wwu~w~eay 

Orir~w 

KY~y@ dy)i 

ti~wi.arwrrr rw~w,rnwr~.www ww~"~iw .+wrwwr. .~wr+~ 

"riw FiY~~ a "~r~w~~A~h" w» ar wwrwwww 

K~w~a ~yO 

M~~Arr".o~rrwM WWy~r~w WMYY+ wWer+w~errprt~" 

t1RU~hdrYIa1 qOdArI "doe 

1~. q" its ~p1~~0"t a~ 14 w" 1"l~ilriiiroi a~ 1! ad7t 

,wp.y. OU ~, CIdaU"rT006 oNbOr7006 

or~r,~r~+r" wwr rw r .,w~w nrww~.r .w~w~r~rwwrnr.r 

~k~~wwwr 

Am ~r ago~ on rwwr~rrrw rwl wr ~r+~~wwwr~wrw~~u .wrwawuY 

arw~ r w w hrnwro ~. ~~.+ rwra~rr~ww+wawr~wwr.r.rrwn 

wrr wrwr ~~ do au. wrri.0rwrew~av. 

rrawrM 
p. 6 

page 1 of 41 

PUBLIC COPY 

ALGA, GROWTH INHIBITION TEST 

Author: 

Sponsor: Testing facility: 
page 2 of 41 

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 AND METHODS 11 

- TEsT ITEM 

- TEsT srEcms 

~Z r `~, t~ . 11 

12 

13 

13-14 

15 

16 

- PRE-TREATMENT OF THE TEST ITEM 

- ExposuRp coNDrnoNs 

- EXPREssION OF BIOLOOICAL RE3ULTs 

- APPLMD SOPS 

RESULTS 17-30 

~ ~~ ; ~ , 

TES 

::. . - 

ARCHIVATION OF DOCUMENTS AND T ARTICLEBvF-- 31 

, 

PUBLIC COPY 
Study number ~ 

Test Item : 

CAS number : 

CONTENTS (continued) 

page 3 of 41 

page 

FIGURE 1: CONCLNTRATiON-EFFECT CURVE (EeC VALUES) 32 

FIGURE 2: CONCENTRATION-EFFECT CURVE (EC VALUES) 33 

FIGURE 3 - 4: GROWTH CURVES 34-35 

ANNEX 1: NUTRIENT MEDIUM PRE-CULTURES AND TEST CULTURES 36 

ANNEX 2: EQUATIONS 37-38 

ANNEX 3 WILLIAMS MULTIPLE SEQUENTIAL T-TEST PROCEDURE 39-40 

GLP CERTIFICATE 41 

PUBLIC COPY 
Study 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 COPY 
page 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 of these inspections 

at the dates mentioned below. 

Date of plan inspection: Date of S report: 

specification study plan 

2007-02-02 

2007-02-02 

Date of A inspection: Date of S report: 

process-based inspection 

SOP 00001 

2006-08-23 

2006-08-23 

Date of report i nspection: Date of report inspection : 

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 COPY 
Study 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 subspicatm over 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 

The cell densities 

in water.

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 COPY 
p. 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 COPY 
p. 14 

page 9 of 41 

Study number " 

Test Item 

CAS number : - 

INTRODUCTION 

and the results obtained during a study This report contains a 

to investigate adverse =Vim" the growth and the growth rate of a 

population of the planlctanic water algal species Desmodes»nw subspicatrrs. 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 COPY 
Study 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 of this 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 of water solubility, respectively . All 

effect data are expressed on the basis of growth [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 item will lead to a 

certain inhibition of algal 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 

pH is 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 item shall 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 COPY 
Study nnmber 

Test Item . 

CAS number Iq : --

Teat Item 

CAS number 

CAS preferred name 

Chemical name 

Article number 

Batch 

Empirical formula 

MATERIAL AND METHODS 

. 

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 COPY 
Study 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 m72 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 from the 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 COPY 
Study numbe 

Test Item 

CAS number 

MATERIALS AND METHODS 

rmdmment of the test item 

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 item were 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 item were 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 of algal 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 item were 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 of alga inoculum. 

Exposure conditions 

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 400 to 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 m72 x 9", or an equivalent 

range of 6000 to 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 COPY 
Study 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 per millilitre 

Test concentration/s 

(nominal) : 0.9, 1 .9, 4.3, 9.4, 20.7, 45.5 and 100 mg/L; also 

100 mg/L filtered solution 

Method of administration : stock "solution" 

Duration of exposure : 72 hours 

Criteria of effects : 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 COPY 
Study namber page 15 of 41 

Test Item 

CAS number 

MATERIALS AND METHODS 

Eznression of bioloai alres 1ts 

" 

" 

" 

Cell density measurements in the test and control cultures are tabulated 

according to the concentration of test item and 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 COPY 
Study nnmbe 

CAS number : 

page 16 of 41 

Test Item 

MATERIALS AND METHODS 

ADDIied 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 pH measurement 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 COPY 
Study 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 of cell growth . 0.0 

Growth rate (r) 

Specific growth rate (0) . 1 .59 

Percentage inhibition of cell growth rate . 0.0 

pH values 

Analysi1 

1 

Oh 

8.2 I 

72 h 

75 I 

PUBLIC COPY 
- page 18 of 41 

Study number 

Test Item 

CAS number 

RESULTS* 

Test item concentration [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 of cell growth . -0.2 

Growth rate (r) 

Specific growth rate (0) . 1 .60 

Percentage increase of cell growth rate . -0.6 

pH values 

Analysis 

I 

Oh 

8.2 

j 72 h 

8.5 I 

PUBLIC COPY 
Study num"'---J 

Test Item 

CAS nnmber : 

RESULTS 

Test item concentration [mg/L] : 1,9 

page 19 of 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 of cell growth rate . 10.4 

pH values 

AnItysi. I 0b 

8.1 

l 

I 

72 h 

8.4 I 

PUBLIC COPY 
Study 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 = 5000 cells/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 

pH valuea 

Analysis I oe 

8.0 

I 

I 

72 h 

720 I 

PUBLIC COPY 
Study 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 of cell growth rate 

F pH valum I 

h 

8.o 

Analysis 

I 

page 21 of 41 

1 .39 

12.5 

72 h 

727 I 

PUBLIC COPY 
-- page 22 of 41 

Study nnmbeli~ 

Test Item 

CAS number : 

RESULTS 

Teat item concentration [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 

pH values 

Analysis 

I 

O _ h 

80 

1 .44 

9.7 

72 h 

8.1 I 

PUBLIC COPY 
Study nnmbeAl 

Test item 

CAS number : 

RESULTS 

Test item concentration [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 of cell growth . 74.3 

Growth rate (r) 

Specific growth rate (0) 

Percentage inhibition of cell growth rate 

pH value; 

Analyab I Oh 

8.1 I 

72 b 

70 

U 

V I 

Ow"'LIC CO 

1 .04 

34.5 

I 
Study numbe 

Test Item 

CAS number 

RESULTS 

Teat item concentration [mg/L] : 100 

page 24 of 41 

. 

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 of cell growth rate . 43.7 

pH values 

Analysis I 

I 8.1 

I 

I 

72 h 

8.0 I 

PUBLIC COPY 
Study nnmbe 

CAS number : 

page 25 of 41 

Teat Item 

---- 

RESULTS 

Test item concentration [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 of cell growth . 0.0 

Growth rate (r) 

Specific growth rate (0) . 1.60 

Percentage inhibition of cell growth rate . 0.0 

An 

pH values 

alysis I Oh 

8.1 

I 

I 

72 h 

8.4 I 

PUBLIC COPY 
Study number : 

Teat Item 

CAS number 

page 26 of 41 

RESULTS 

Test item concentraNon [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 of cell growth . 29.3 

Growth rate (r) 

Specific growth rate (0) 

Percentage inhibition of cell growth rate 

pH values 

Analysis 

I 

Oh 

8.2 

1.44 

10.0 

I 

I 

72 h 

737 I 
page 27 of 41 

Study number :-

Teat item 

CAS nnm . COPY 

SUMMARY OF RESULTS 

Mum cell deaft during the tat 

Tat ihm coaceatrstloo 

[nWLl 

Cell deedty [=111/10L1 

(initial cell dene#y = 5000 cxlle/mL) 

24Y 4Sh 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 7 
Study number 

Test Item 

CAS number : 

page 28 of 41 

PUBLIC--- COPY 

SUMMARY OF RESULTS 

Mean cell denrity during the teat 

Tat item concentratlon 

[~1 

" 

Cel1 deadty [celWmL] 

(untial cell densaty = 5000 cells/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 10.0 
p. 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 of the test suspension after 72 h revealed that algal cells were in 

free suspension. Very few were observed attached to flocs or lumps of aggregated test item 

This indicated that "trapping" of algae in test item did not contribute significantly to growth 

inhibition . 

A blank suspension of the test item was 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 itnm pore size). The clear filtrate was then 

used in the algal test and resulted in 10.0% inhibition of growth rate compared to control. 

PUBLIC COPY 

/19 
Study number 

Test Item 

CAS number : 

page 30 of 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 medium for 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 CO 
Study numbe 

Test item 

page 31 of 41 

CAS number : 

ARCHIVATION OF DOCUMENTS AND TEST ARTICLES 

Study plans, raw data and study reports are archived at the GLP archives of the 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 item used where sampling has not been 

performed 

PUBLIC COP 
p. 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) of the 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 COPY 
Study 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 of the probit analysis: Selected effective concentrations (ECx) of the test item and 

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 - 

so40 . 

ao 

:o 

to 

1o 10 0 100 0 10 1 00 1000 00 Concentration [mg/1.1 

0 Date 

IYIIOMOq 

"G% -CL 

PUBLIC COPY 
Figure 3: 

Growth curves 

s 

.; 100 

low , 

24 48 n 

tcWAMI -O-a9 mWl. --w-4a ny#L -rr-9.4=Wt -F-zo.7mWrt. -t4s.s m@Ot. i00 mOt. 

PUBLIC COPY 
p. 40 

Study number 

Teat Item 

CAS number . 

Figure 4: 

Growth carve: 

1 

1000 

100 

10 

1 

0 

hoan 

-~Cootol (lae eaeomanlios n hr SYwed ad tlom wYY 1hmdm --F 100 (mmfed Sllemd) 

page 35 of 41 

PUBLIC COPY 
p. 41 

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 

CaC12 x 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 80 mg/1 80 pg/1 

NazEDTA x 2 HZO 100 mg/1 100 As/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 6 H20 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 of the 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 

/ ;- 6 
p. 42 

Study number 

Test item 

CAS nnmbe . 

ANNEX Z/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 of the test (7Z 6). 

No is the nominal initial cell density, 

Ni, N=, Ns is the measured cell density at time ti, t2, ts . 

From the 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 : 

ANNEX 3/1 

Determination of NOEC and LOEC based on growth (b) according to 

Williams 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; °/.MDD: minimum detectable 

difference to Control (in percent of Control); t: sample t; t* : critical t for Ho: p1= IL2 

=. . . = * the differences are significant in case (1 > jt*j (The residual variance of an 

ANOVA was 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 COPY 
ANNEX 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 

ANOVA was applied; df = N - k; N: sum of treatment 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 NOEC of 0.900 mg/L is suggested by the prograaL 
p. 46 

page 41 of 41 

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On~i~~~1~YA6s 
STUDY FOR THE SIGN SENS1TiZATION EFFECT 

IN GUINEA PIGS 

(Guinea Pig Maximization Test according to Magnuason and Kligman) 

PUBLIC COPY 

Study Completion Date: March 30, 2007 

Page 1 of 25 
GLP 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 COPY 
INDEX 

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 . Speckm and 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 

PUBLIC COPY 

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 AND ASSESSMENT 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 

PUBLIC COPY 
p. 51 

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 

PUBLIC COPY 

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: 

PUBLIC CO Y 
p. 53 

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 item concentrations : 

First induction : 23.896 

Second induction: 23.8% 

Challenge: 23.896 

The test item was 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 item group and the control group. 

In summary, under the conditions of the maximization test and with respect to the 

evaluation criteria the test item therefore exhibits no skin-sensitization potential. 

PUBLIC COPY 
4. 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 are 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 COPY 
5_.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 CO 
8. MATERIAL AND METHODS 

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 COPY 
p. 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 COPY 
p. 58 

6.3 . Animal Care 

' 63. 1 . Animal Accommodation 

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 Rooms 

All animal§ used in this study were kept in the same animal room, one In which guinea pigs from other 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. Disinfection 

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. KILLGERM Roach Traps®, a cockroach trap which uses no pesticides, was placed in the animal room. 

PUBLIC COPY 
8.3 .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 ` p e n ki 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 item formulation 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 item formulation 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 Formulation 

15 

PUBLIC COPY 

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 item group 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 row were 

made on the left and the right side of the spinal column. Hypoallergenic patches 

(2 cm x 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 Item group: 250 m 

06W0 

ml vaseline 

b) Control group: 0.5 mi vaseline 

At the end of the 24-hour exposure period, the remaining test item was 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 item group: 250 .0 ml vaseline 

b) Control group: 0.5 ml vaseline 

At the end of the 48-hour exposure period, the remaining test Item was 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 item was 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. 

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6.5 .7. Evaluation of the Results 

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 item group 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). 

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7. RESULTS 

7.1 . General Examinations 

Appearance and behaviour of the test item group were not different from the 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 item group 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 item group: 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 item group 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 item group (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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p. 66 

0"0 20 . 

400"-N"~) 

8. DISCUSSION AND ASSESSMENT 

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 item concentration . 

The test item was 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. 

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9. 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 248 Vol. 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 

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10. 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 . In dividual Range-finding (Pilot) Test Results 

Dose-ranoe 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 item or 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 item or 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 item patch Control patch 

Animal 48h# 72h# 48h# 72h# 

No, Contr ol g roup 

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 h em group 

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 group 

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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Acak b0=ieNy in th! rat 

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p. 72 

Study CamPietton Date: Decemba 4, 2006 

Page 1 of 27 
GLP Compliance Statement 

This stndy was conducted in compliance with the OECD Principles of Good 

Laboratory Practice as revised in 1997 [ENV/MGCHEM(98)17] and with the 

revised German Principles of Good Laboratory Practice according to Annex 1 

German Chemicals Act (Buadesgesetzblatt Part 1, No 40, issued June 27, 2002). 

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Aelv.,!z affG 

no 
p. 74 

Table of Contents 

l . Signatures.-.. .N.tN.NNNNN.N.N.NNNNN.N. NNNN.NNNNNNNN.NNNNN S 

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 of the 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 of the 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 of Abbreviations 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-term studies 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 

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(~M 

Dste: 

no results of ft 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 of anknalt wilh WW* of tooddly I Ift number of W*rAft LOW Par 

Band an the present inv~ to be regarded to have 

tha following LD50 values : - 

opp"p 

LD50 rat, male : > 2000 mg/kg body weiQht 

rat, hnale : > 2000 m&ft body weight 

3o it is regarded ac nontoxic aft dermal applicadon. 

(OH3 Category Sheerlassifled analogous OECD draft guideline 434) 

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'/ 

Groups of s male and s female wislar rob received s single dermal dose of 

2000 mift body weqft of to test compound applied amocclusively for 24 

lwum 

A dose of 2000 m8/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 with the OECD-(inidoline for Taft of Chemicals 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 the principles 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 of the 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 of the Council of the European Communities of 27 Jian% 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 of a retention sample is in the 

responsibility o aponsor. 

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4. 

9 

Identitieation and Responsibilities 

4.1 Identification of the Study 

Study No. 

4.2 ResponsibiUtier 

Head of Department 

Head of Test Facility 

Arcbving No 

Heed of Quality Assurance U ]jj 

Study Director 

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s. 

19 

Material and MeMods 

The "Acute Dermal Toxicity" descn'bod in OECD guideline 402 was used for 

judgment on the acute toxicity of the test substimce . For classification according to 

the Globally Harmonized System of Classification and Labeling of Chemical` 

((1H3) do OECD draft guideline 434 was used. 

5.1 Test Compound 

Test compound 

Chemical name 

: 9S. used for calculations) 

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

Conflrmadon of the identity of the tat compound was performed. 

Hatch no. 

Content of test aubatanov 

Appearanoe : black, povvdery to coarse-grained 

Storage : room tomperattra 

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p. 82 

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&2 Exposure Prmedurs 

One day before the stat of the trmftw the back aod flub of the rats were shoam 

(~proximately 10% of the 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') of a 

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 

(8 cm x 23 cm) and additionally covered with a "Lomir biomedical Ino rat jackct", 

which was connected with a 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 of stability was 

not performed . 

5.3 Application Does md Exposure Period 

Depending on the body weight of the 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 20.0 om' 22.1 - 24.e 

2000 20.o cm' 20.5 - 22.0 
p. 83 

~ PUBLIC C 

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 of this strain have 

been toxicological studies for many years . 

Historical data on their phyalology and spontaneoas alterations is available . The 

state of health of the brooding colony is routinely spot-c>mclced for the main specific 

pathogens. The results of then examinations are archived 

At start of the study the animals were nulliparous and non-pregnant and free of all 

clinical symptoms or diseases. T1re seelimatisatlon time in the animal room was at 

least S days. 

Body weights at start of study: 

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 random list 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 10 changes per hour 

Lightl Dark cycle : 12 hours rhythm 

Occasional deviations from these standards oceurred, e.g. during cleaning of the 

animal room. They did not have any apparent influence on the outcome of the 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 of the animals were placed on racks . The wood granulate wu randomly 

checked for contaminants at regular intervals and the results have been stored at the 

analyses yielded no evidence of any adverse 

effects on the aim of the 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 of the 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 libitum from polycarbonate 

bottles. 
p. 85 

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

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

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

Decree in the cun+ent version). The results of the analyses have been sto 

avsilable dats yielded no 

evidence of any sim of the study. The food was available 

from racks in the lid of the 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. Killgecm Roach Trap, Killgerm C3mbH, 41460 Neuss, Germany). The contact 

of the 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 of Chemicals; Section 4: Health Effects, No. 402 -

"Acute Dermal Toxicity"; adopted: 24 February, 1987 ('Ilurd Addendum to the 1981 

OECD Guidelines for Testing of Chemicals, OECD Publication Service, Paris 

1987); Annex V, Part H.3. (acute toxicity [dermal]) to Directive 67/548/EEC of the 

Council of the 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 of at 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 day were 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, a further observation 

intervals are given in days. Tbe duration of the symptoms and the times of death are 

given relative to the time of application 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 of death will be 

talsen as the first occurrence of a symptom. In the 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 of the animals was checked weekly until the and of the 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 of the 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 of the 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-1000 A (4x, operating system RTE-A 6.2) 

" HP-3000 series 900 (lx, operating system heErlX 5.5) 

" HP-9000 series 200 (lx, operating system HP-UX 11.0) 

Software : Data was stored on HP-3000 in an HP 'IurboImmagdXi. Database. 

5.9 Calculation of the LDSO 

An assessment of the LDSO was made based on the appUed dose and doee-response 

ctuve, respectively . 
p. 88 

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6. Reaulb 

6.1 DowRespoase Table (LM) 

The reaulta of the 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 of animala with signs of baoddty I bohl number of animals wad per 

group 

LDS0 male : > 2000 mqft bw. 

female : > 2000 mg/!cg bw. 

(aHS cateM 5/unclassified ralopom OECD draft guideline 434) 

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 Weight gain of three females in the 

Srst week is assumed to be caused by the atrees of the 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 of the study revealed no particular findings (see 

aPPouft. Pap 25). 
19 PUBLIC COPY 

7. Conclasioe 

the followinQ LD50 values: 

Hased onthe present iovestimptl be regarded to have 

LDSO rat, male . > 2000 mg/lcQ body weight 

rat, fanale : > 2000 mg/kg body weight 

3o it is regarded as nontoxic aRet decmal application. 

(GH3 Category S/unclassi8ed melogoos OECD dnft guideline 434) 
p. 91 

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S. Appendix 

List of Abbreviations Used In the Appendix 

In addition to the abbreviations for the basic units of the 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 of death 

ta treatment ares 

' minute 

°A percent 

fr.cnale 

a male 
p. 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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p. 96 

25 

1ndividual nacroscopic findinOs 

Alt findings 

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

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

anisrl tisn / type finding 

no. of death 

.. . .. . . . . ,- ..--t---------- "---------------------------------------

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

1 I 15d III 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 

PUBLIC COPY 

10 1 iSd / E I General observations 

1 I no pathological finding 
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aftee ord adsi~bh_tioi 

Study Compledon Deoe: Decaaber 4, 2006 

Pap 1 of 28 
Except 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 of the 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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