TS-CKHI02

Attachment #10

Environment Effects Data on the New Chemical Substance

Acute Fish Toxicity Test

## LAB Resea rch Ltd.

Address: 8200 Veszprém, Szabadságpuszta

Phone: +36 88 545 300

Fax: +36 88 545 301



### FINAL REPORT

ACUTE TOXICITY

TEST WITH

Polymer

ON ZEBRAFISH

( Brachydanio rerio )

Date of Final Report: 04 April 2011 

### STUDY CODE: 10/339-009H

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## LAB GENERAL INFORMATION

STUDY TITLE : Acute toxicity test with Polymer on Zebrafish ( Brachydanio rerio )

TEST ITEM :

SPONSO R :

TEST FACILITY : LAB Research Ltd.

Address: H-8200 Veszprém, Szabadságpuszta Hungary

Phone: +36 88 545-300

Fax: +36 88 545-301

STUDY DIRECTOR : István Ágh M.Sc.

Phone: 36 88 545 344

e- mail: istvan.agh@labresearch.hu

QUALITY ASSURANCE : Ramóna Heiderné Grób B.Sc.

TECHNICAL STAFF : Ecotoxicological staff

START OF EXPERIMENT : 21 February 2011

EN D OF EXPERIMENT : 25 February 2011

BASIS OF STUDY:

– OECD Guideline for Testing of Chemicals, No. 203, "Fish, Acute Toxicity Test", adopted July 17, 1992

– EPA Health Effects Test Guidelines, OPPTS 850.1075

– Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restrictions of Chemicals (REACH), Annex Part C, C.1 (published in the Official Journal o f the European Union L 142 of 31 May 2008)

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## LAB CONTENTS

Page

1. SUMMARY 7

2. INTRODUCTION 8

3. MATERIALS AND METHOD S 8

3.1. TEST ITEM AND CONTRO LS AND VEHICLE 8

3.2. EXPERIMENTAL ANIMALS 9

3.3. ENVIRONMENTAL TEST CONDITIONS 10

3.4. DESCRIPTION OF THE TEST PROCEDURE 10

3.5. OBSERVATIONS 11

3.6. ANIMAL WELFARE 11

3.7. PERMISSION OF THE IACUC 11

3.8. STATISTICAL EVALUATION 11

3.9. ANALYTICAL MEASUREMENTS 11

3.10. ARCHIVES 12

3.11. VALIDITY CRITERIA OF THE STUDY 12

3.12. DISTRIBUTION OF THE FINAL REPORT 12

3.13. REFERENCES 13

4. RESULTS 14

4.1. VALIDITY 14

4.2. MORTALITY DATA 14

4.3. SUB

- LETHAL EFFECTS 14

4.4. BODY WEIGHT 14

5. CONCLUSION 15

APPENDICES 16

Data of Test conditions 1 o f A ppendix 1
Body Length of Test Fish 1 of Appendix 2
Copy of the GLP Certificate 1 of Appendix 3

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## LAB 1. SUMMARY

The a cute toxicity of Polymer was assess ed with acute fish toxicity

test o n Zebrafish ( Brachydanio rerio ), over an exposure period of 96 hours.

Toxic effects were not observed during the preliminary concentration range

- finding test therefore a limit test was performed in the main experiment usi ng only one test concentration at the limit of solubility of the test item in the test medium (100 % v/v saturated solution) and one control group

. Because the test item is extremely poorly soluble in water or in any practicable organic solvent, and in add ition no analytical measurement is possible for the determination of stability in the test medium over the test duration, the test item solution used in the test was prepared by a method which ensured that the test was carried out at the limit of solubility of the test item (limit test concentration) during the whole test period.
One aquarium was used in test group and one in the control group. Both aquaria comprised seven fish and twenty litre test solution.
The number of mortalities and any sub

- lethal eff ect were determined 3, 6, 24, 48, 72 and 96 hours after the start of exposure.

All validity criteria were met during this study.

Under the conditions of this acute fish toxicity study on zebrafish ( Brachydanio

rerio ) the observed endpoints for the effec t of Polymer were the followings:

The 24h, 48h, 72h and 96h LC 50 value: > 100 % v/v saturated solution

The 96h NOEC : 100 % v/v saturated solution

The 96h LOEC : > 100 % v/v saturated solution

The 96h LC 100 value: > 100 % v/v saturated solut ion

Based on the results of this study, the test item Polymer had no toxic effect at aquatic saturation on fish; the LC 50 results and the LOEC are higher than the solubility level of the test item in the test medium.

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## LAB 2. INTRODUCTION

The objective of this study was to determine the median lethal concentration (LC 50 value) of the test item in acute toxicity test on zebrafish ( Brachydanio rerio ).

F ish were exposed to aqueous test media containing the test item at one concentration at the limit of solubility to demonstrate that the test item has no influence on test animals up to at least this test concentration level

. The test method of application and the test species zebrafish ( Brachydanio rerio ) are recommended by the test guidelines.

3. MATERIALS AND METHOD S

3.1. TEST ITEM AND CONTRO LS AND VEHICLE

3.1.1. Test Item

Name:

Chemical name:

Lot Number: Purity: 98.6 % Description: white / waxy solid Expiry date: 23 August 2012 Storage condition: Room temperature (15 -30 °C); humidity (40

- 70%);

in the dark Safety Precautions: Routine safety precautions (gloves, gog gles, face mask, lab coat) for unknown materials will be applied to assure personnel health and safety

Manufacturer: see Sponsor

The test item of a suitable chemical purity was supplied by the Sponsor. All precautions required in the handling and disposa l of the test item were outlined by the Sponsor. These documents are part of the raw data. Identification of the test item was performed in the Central Dispensary of LAB Research Ltd

. on the basis of the information provided by the Sponsor.

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## LAB 3.1.2. Dilut ion and Preparation of Testing Solutions

Because the test item is extremely poorly soluble in water or in any practicable organic solvent, the test was carried out in a saturated solution of the test item in water (limit test concentration) during the who le test period in a recirculation system. Due to the extremely poorly water solubility, no analytical measurement was possible for the determination of concentration or stability in the test medium.

The method is based on the elution of the test substance with the test medium from a column, which is charged with an inert support material (e.g. glass beads or quartz sand) mixed with an excess of the test substance. For this method aquarium water is

used as eluent.

Small particles of test substance (with a relatively high surface area) were mixed with support material (glass beads w ere used in this test). This mixture was poured into the

column and then filled with aquarium water . The column was connected to a re

circulating pump. The flow through the column was started approximately 10 hours before the introduction of the fish (to achieve the constant saturated solution) and run over the test period. The flow rate of the water pumped through the column filled with

test item allowed a theoretical recirculatio n of the whole tank volume approximately every 12 minutes, so 10 hour represented a recirculation of approximately 50 times.

3.1.3. Untreated Control

The dilution water (c irculated and filtered tap water ) was used without of addition of
the test item.

3.2. EXPERIMENTAL ANIMALS

Species: Zebrafish ( Brachydanio rerio )

Source: “Black Molly” Fish Farm, Veszprém, Hungary
Justification of species: Zebrafish ( Brachydanio rerio ) is one of the convenient species for acute fish toxicity test.
Number of animals: seven fish per test group
Body length of animals: 2.7 – 3.2 cm Food and Feeding: The fish were not fed during the test

Acclimatisation: >12 days

Animal health: Fish were bred in a well

- known fish farm, under disease

and parasite

- controlled conditions . Fish were observed during the acclimatisation and test periods, the fish were healthy.
There was no mortality of the population for seven days

before the test.

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## LAB 3.3. ENVIRONMENTAL TEST CONDITIONS

The water temperature, oxygen concentration and pH were mea sured at the start of the test and daily thereafter

.

The test temperature was between 22.0 and 22.3 °C.

The dissolved oxygen concentration was in the range of 91 – 93 % of the air saturation value at the temperature used.

The pH was in the range of 8.08 – 8.20.

The hardness of the dilution water was determined as 249 mg/L (as CaCO 3 ).

The light-dark cycle during the test was 16 hours light and 8 hours darkness.

The test conditions above are detailed in Appendix 1.

3.4. DESCRIPTION OF THE TEST PROCEDUR E

The test duration was 96 hours. One aquarium was used for test group and for the control group respectively

. Both aquari a comprised 7 fish and 20 L test solution. The

animals were not fed during the test

. The loading of the test aquaria was less than 1.0 g fish / L test solution at the start of the experiment.

The choice of the test concentrations was done on the basis of the results of the preliminary range -finding test.

3.4.1. Preliminary Range -Finding Test

Three fish were exposed to a single test concentration at aquatic saturation and one control in order to demonstrate that the test item had no influence on fish up to at least this test concentration (100 % v/v saturated solution). The test solution was prepared using method as described in section 3.1.2..

No mortality or any toxic effect was detected in the treated concentration (100 % v/v saturated solution) and in the control groups during the 96 h exposure period.

3.4.2. Concentration Levels Investigated in the Main Test

Because toxicity w as not observed during the preliminary test, only one test
concentration at aquatic saturation and one untreated control were tested in a limit test

.

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## LAB 3.5. OBSERVATIONS

The observations of fish were carried out in the following intervals: at 3, 6, 24, 4 8, 72

and 96 hours. Mortality and any sub

- lethal effect were determined in each observation period.

The test conditions (pH, temperature, oxygen saturation) were examined daily during

the test.

The body weights of the fish were recorded before the introduction of fish

. The body weight of 7 fish per aquarium was registered . T he loading of the aquaria was calculated on the basis of these body weights (g fish/litre testing liquid).

3.6. ANIMAL WELFARE

After the experiment, all fish were humanely killed. Eu thanasia for all animals was applied by an overdose of an aqueous solution of 200 mg/L Tricaine Methanesulphonate (MS 222; CAS No.: 886 -86- 2). This technique is considered to provide a humane euthanasia by direct depression of the brain and vital centers.

3.7. PERMISSION OF THE IACUC

The conduct of the study was permitted by Institutional Animal Care and Use Committee (IACUC) of LAB Research Ltd. Date of IACUC approval: 02 February 2011

3.8. STATISTICAL EVALUATION

No statistical analysis was perfor med because of the lack of toxic effects.

The LC 50 , NOEC

, LOEC and LC 100 were determined directly from the raw data.

3.9. ANALYTICAL MEASUREMENTS

Due to the extremely low aquatic solubility of the test substance, analytical

determination of its concentr ation in the test medium wa s not possible. A system was used to assure that a saturated solution was used throughout the study by recirculation of the aquatic media through a column containing the test item. To demonstrate that

the test solution was satura ted during the whole exposure period, visual assessment of

the test substance

- support material mixture was undertaken at the beginning and end

of the test. These observations showed that test item was present throughout the study in the recirculation syste m at ~50 recirculations per 10 hours over a high surface area of test item; this is considered sufficient evidence that the solution was saturated during the study.

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## LAB 3.10. ARCHIVES

The study documents:

- study plan and amendment,

- all raw data,

- sam ple of test item, - study report, and any amendments, - correspondence

are stored according to the Hungarian GLP and to the LAB Research Ltd. SOP-s in the archives of LAB Research Ltd. 8200 Veszprém, Szabadságpuszta, Hungary.

After the retention time has elapsed all the archived materials listed above will be returned to the Sponsor or retained for a further period if agreed by a contract.

Otherwise the materials will be discarded

.

3.11. VALIDITY CRITERIA OF THE STUDY

- Constant conditions should be maintained in this procedure.

- The mortality in the controls should not exceed 10 % at the end of the test (or one

fish if less than ten are used). In case of limit test, no mortality should be occurred.

- The dissolved oxygen concentration (throughout the t est) has to be at least

60 % of air saturation value.

3.12. DISTRIBUTION OF THE FINAL REPORT

Sponsor: 1x copy, bound 1x copy, unbound

1x PDF file

Archive: 1x original, bound

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## LAB 3.13. REFERENCES

1. OECD Principles of Good Laboratory Practice, adop ted by Council on 26 th
November 1997 [C(97)186/Final], Environment Directorate, Organisation for Economic Co-operation and Development, Paris 1998.

2. Hungarian Good Laboratory Practice Regulations: 9/2001 (III. 30) EÜM

- FVM joint

decree of the Minister of Hea lth and the Minister of Agriculture and Regional Development which corresponds to the OECD GLP, 1997

3. Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restrictions of Chemicals (REAC H ), Annex Part C, C. 1 (published in the Official Journal of the European Union L 142 of 31 May 2008)

4. EPA Guideline 712 -C-96- 118: OPPTS 850.1075, "Fish Acute Toxici ty Test, Freshwater and Marine" April 1996

5. OECD Guideline for Testing of Chemicals, Section 2, No. 203, "Fish, Acute Toxicity Test", adopted July 17, 1992.

6. Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3. 1.2 Media preparation methods, Direct addition. OECD Series on Testing and Assessment No. 23, Paris September 2000.

7. Guidelines for studies on the new chemical substance as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973, amended 2009 under the reference of YAKUSHOKHATSU No. 1121002, SEIKYOKU No.2 and KANPOKIHATSU No. 021121002 and partially amended 2006 as the joint ordinance of The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE)

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## LAB 4. RESULTS

4.1. VALIDITY

The validity criteria were within acceptable limits and therefore the study can be

considered as valid. Evi dence that the test item was present at saturation during the study is considered to be sufficient.

4.2. MORTALITY DATA

C umulative mortality data from the exposure of zebrafish to the test material during

the definitive test are given below in Table 1.

There were no mortalities in 7 fish in the control group or in 7 fish exposed to a test

concentration of 100 % v/v saturated solution

.

Table 1: Cumulative mortality data in the definitive test

Test Group Mortality rate of treated fish (Dead fish/Treated fish)

3h 6h 24h 48h 72h 96h

Control 0/7 0/7 0/7 0/7 0/7 0/7

100 % v/v saturated solution 0/7 0/7 0/7 0/7 0/7 0/7

4.3. SUB

- LETHAL EFFECTS

No sub- lethal effects were observed in the control and test group for a period of

96 hours.

4.4. BODY WEIGHT

The body weight of 7 fish was weighed at the start of the test. The measured and

calculated data are listed below in Table 2.

Table 2: Measured and calculated data of bodyweight

Test Group Measured weight of 7 fish (g) Calculated mean weight of 1 fish ( g) Loading of testing aquarium (g fish/L testing liquid)

Control 4.05 0.58 0.20

100 % v/v saturated

solution 4.09 0.58 0.20

There was no considerable difference observed concerning body weights between the groups.

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## LAB 5. CONCLUSION

The acute toxicity of Polymer was assessed with acute fish toxicity

test on Zebrafish ( Brachydanio rerio ), over an exposure period of 96 hours.

One concentration at the solubility level of the test item in the test medium ( 100 % v/v saturated solution) plus a n untreated control was tested in a limit test.

All validity criteria were met during this study.

Under the conditions of this acute fish toxicity study on zebrafish ( Brachydanio

rerio ) the observed endpoints for the effect of Polymer we re the followings:

The 24h, 48h, 72h and 96h LC 50 value: > 100 % v/v saturated solution

The 96h NOEC : 100 % v/v saturated solution

The 96h LOEC : > 100 % v/v saturated solution

The 96h LC 100 value: > 100 % v/v saturated solution

Based on the results of t his study, the test item Polymer had no toxic effect at aquatic saturation on fish; the LC 50 results and the LOEC are higher than the solubility level of the test item in the test medium.

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## LAB 

## APPENDICES

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## LAB APPENDIX 1

DATA OF TEST CONDITIONS

Table 3: Temperature (°C)

Test Group Measuring

0h 24h 48h 72h 96h

Control 22.2 22.2 22.0 22.3 22.0

100 % v/v saturated solution 22.2 22.1 22.1 22.3 22.0

Table 4 : Dissolved oxygen concentration (% of the air saturation value)

Test Group Measu ring

0h 24h 48h 72h 96h

Control 92 92 93 92 93

100 % v/v saturated solution 91 92 92 92 92

Table 5: pH

Test Group Measuring

0h 24h 48h 72h 96h

Control 8.12 8.10 8.14 8.10 8.16

100 % v/v saturated solution 8.17 8.08 8.12 8.09 8.20

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## LAB APPENDIX 2

BODY LENGTH OF TEST FISH

Table 6: Body length of test fish at the end of the test

Test Group Body l ength [cm]

1 2 3 4 5 6 7

Control 2.8 3.0 3.0 2.9 3.0 3.2 2.9

100 % v/v saturated solution 2.9 3.0 3.2 3.1 3.1 2.7 2.8

Remark: At the end of th e test, body length of test fish were measured in order to check their compliance with the size range recommended for the species by the test guideline [ Commission Regulation (EC) No 440/2008; Annex Part C, C.1]

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## LAB APPENDIX 3

COPY OF THE GLP CERTIFICATE