# FINAL REPORT

Study Title



# ASSESSMENT OF ACUTE ORAL TOXICITY WITH

CASTOR OIL, SULFATED , SODIUM SALT, 75%

IN THE RAT (UP AND DOWN PROCEDURE)

Author

A.H.B.M. van Huygevoort, MSc.

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 503339

Substance 205003/A

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503339

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### 1. CONTENTS

1. CONTENTS

................................................................................................ ...................................... 2

2. STATEMENT OF GLP COM PLIANCE

............................................................................................. 3

3. QUALITY ASSURANCE ST ATEMENT

............................................................................................ 4

4. SUMMARY

................................................................................................ ........................................ 5

5. INTRODUCTION

.............................................................................................................................. 6

5.1. Preface

................................................................................................ ..................................... 6

5.2. Aims of the study

...................................................................................................................... 6

5.3. Guidelines

................................................................................................ ................................ 6

5.4. Storage and retention of records and materials

....................................................................... 7

6. MATERIALS AND METHOD S

.......................................................................................................... 8

6.1. Test substance

......................................................................................................................... 8

6.1.1. Test substance information

............................................................................................. 8

6.1.2. Study specific test substance information

...................................................................... 8

6.1.3. Test substance preparation

............................................................................................ 8

6.2. Test system

.............................................................................................................................. 8

6.3. Animal husbandry

.................................................................................................................... 8

6.4. Study design

............................................................................................................................ 9

6.5. Treatment

................................................................................................ ................................ . 9

6.6. Observations

............................................................................................................................ 9

6.7. Electronic data capture

............................................................................................................ 9

6.8. Interpretation

.......................................................................................................................... 10

6.9. List of deviations

.................................................................................................................... 10

6.9.1. List of protocol deviations

............................................................................................. 10

6.9.2. List of standard operating procedures deviations

......................................................... 10

7. RESULTS

................................................................................................ ....................................... 10

7.1. Mortality (Table 1)

.................................................................................................................. 10

7.2. Clinical signs (Table 2)

........................................................................................................... 10

7.3. Body weights (Table 3)

.......................................................................................................... 10

7.4. Macroscopic findings (Table 4)

.............................................................................................. 10

8. CONCLUSION

................................................................................................ ................................ 10

T ABLES

Table 1: Mortality

................................................................................................ .................................... 11

Table 2: Clinical s igns

............................................................................................................................. 11

Table 3: Body w eights

............................................................................................................................ 12

Table 4: Macroscopic f indings

................................................................................................................ 12

APPENDICES

APPENDIX 1: TEST SUBSTANCE CERTIFICATE OF ANALY SIS

### Castor Oil, Sulfated, Sodium Salt, 75% Project 503339

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### 4. SUMMARY

Assessment of acute oral toxicity with Castor Oil, Sulfated, Sodium Salt, 75% in the rat (Up and Down

procedure).

The study was carried out based on the guidelines described in:

OECD No.425 (2001) "Acute Oral Toxicity

– Up and Down procedure",

EPA, OPPTS 870.1100 (2002), "Acute Oral Toxicity"

Castor Oil, Sulfated, Sodium Salt, 75% was administered by oral gavage to five female Wistar rats

. A

correction was made for the purity of the test substance (70.7%). The first animal was treated at a

dose level of 2000 mg active ingredient / kg. The absence or presence of mortality at one step

determined the next step, based on the test procedure defined in the guidelines. Based on the results

of this first animal, four additional animals were dosed at 2000 mg/kg. A nimals were subjected to daily

observations and weekly determination of body weight. Macroscopic examination was performed after

terminal sacrifice (Day 15).

No mortality occurred.

Hunched posture and piloerection were noted among the majority of animals between Day s 1 and 3.

The body weight gain shown by the animals over the study period was considered to be normal.

No abnormalities were recorded at macroscopic post mortem examination of the animals.

The oral LD50 value of Castor Oil, Sulfated, Sodium Salt, 75% in Wistar rats was established to

excee d 2000 mg active ingredient /kg body weight

.

Based on these results, Castor Oil, Sulfated, Sodium Salt, 75% does not have to be classified and has

no obligatory labelling requirement for acute oral toxicity according to the:

- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United

Nations (2011),

- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and

mixtures.

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### 5. INTRODUCTION

5.1. Preface

Sponsor Sulfated Castor Oil Consortium, Society of Che mical

Manufacturers & Affiliates

1850 M Street NW, Suite 700

Washington, DC 20036

USA

Study Monitor

(on behalf of the Sponsor) Rodney Boatman, Ph.D.

PCA Services

2704 Trail Wood Drive

Durham, NC 27705

USA

Test Facility WIL Research Europe B.V.

Hamb akenwetering 7

5231 DD 's

- Hertogenbosch

The Netherlands

Study Director A.H.B.M. van Huygevoort, MSc.

Study Plan (in

- life phase) Start : 21 August 2013

Completion : 06 September 2013

5.2. Aims of the study

The objective of this study was to assess the toxicity of the test substance when administered in a

single dose to female rats at one or more defined dosages. Furthermore, the results of the study

allowed the test substance to be ranked according to most classification systems currently in use.

Th is study should provide a rational basis for risk assessment in man.

The oral route was selected as it is a possible route of human exposure during manufacture, handling

or use of the test substance.

5.3. Guidelines

As required by the Dutch Act on Animal Experimentation (February 1997), this type of protocol was

reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee (DEC 05

- 15) as required by the Dutch Act on Animal Experimentation (February 1997). The study procedures

described in this protocol are based on the following guidelines:

Organization for Economic Co

- operation and Development (OECD), OECD Guidelines for Testing of

Chemicals, Section 4, Health Effects, No.425, "Acute Oral Toxicity

– Up and Down procedure", Paris

Cedex , 200 8.

United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS

870.1100, Acute Oral Toxicity. Office of Prevention, Pesticides and Toxic Substances (7101), EPA

712 -C- 02

- 190, 2002.

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### 5.4. Storage and retention of records and m aterials

Records and materials pertaining to the study including protocol, raw data and the final report are

retained in the WIL Research Europe archives for a period of at least 2 years after finalization of the

report. After this period, the sponsor will be contacted to determine how the records and materials

should be handled. WIL Research Europe will retain information concerning decisions made.

WIL Research Europe will retain a test substance sample until the expiry date, but no longer than 10

years a fter finalization of the report. After this period the sample will be destroyed.

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### 6. MATERIALS AND METHOD S

6.1. Test substance

6.1.1. Test substance information

Identification Castor Oil, Sulfated, Sodium Salt, 75%

CAS Number 68187

- 76

- 8

Description Clear amber liq uid

Batch R

- 10135

- 064

Purity/Composition 70.7%

Test substance storage At room temperature in the dark

Stable under storage conditions until 18 July 2014 (expiry date)

See appendix I; Certificate of analysis.

6.1.2. Study specific test substance informati on

Purity/composition correction factor

required Yes, correction factor is 1.41 (70.7% purity)

Specific Gravity 1.049

pH 7.2 at concentration of 5%

Stability at higher temperatures Yes, maximum temperature 100°C,

maximum duration: until dry

6.1.3. Test substance preparation

The test substance was dosed undiluted as delivered by the sponsor. A correction for the purity was

made.

6.2. Test s ystem

Species Rat, Wistar strain Crl:WI (outbred, SPF

- Quality). Recognized by

international guidelines as the recommended test system (e.g. OECD,

EC).

Source: Charles River Deutschland, Sulzfeld, Germany.

Number of animals 5 Females (nulliparous and non

- pregnant).

Age and body weight Young adult animals (approx. 8 weeks old) were selected. Body weight

variation did not excee d +/

- 20% of the sex mean.

Identification Earmark and tail mark

. Health inspection At least prior to dosing. It is ensured that the animals were healthy and

without any abnormality that might affect the study integrity.

6.3. Animal husbandry

Conditions

Environ mental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40

to 70%, approximately 15 room air changes/hour, and a 12

- hour light/12

- hour dark cycle. Any

variations to these conditions were maintained in the raw data and had no effect on the outcome of the

study.

Accommodation

Individually housed in labeled Ma k rolon cages (MIII type, height 18 cm.) containing sterilized sawdust

as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage

- enrichment (Envir o- dri,

Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).

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### Acclimatization period

The acclimatization period was at least 5 days before the start of treatment under laboratory

conditions. Accommodation was as described above except that the animals were group housed in

labeled Ma k rolon cages (MIV type; height 18 cm).

Diet

Free access to pelleted rodent diet (SM R/M

- Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water

Free access to tap water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed

according to facility standard procedures.

There were no findings that could interfere with the study.

6.4. Study design

The toxicity of the test substance was assessed by stepwise treatment of five fema les

. The first animal

was treated at a dose level of 2000 mg/kg. The absence or presence of mortality at one step

determined the next step, based on the test procedure defined in the guidelines. Based on the results

of this first animal, four additional animals were dosed at 2000 mg/kg.

6.5. Treatment

Method Oral gavage, using plastic feeding tubes

.

Fasting Animals were deprived of food overnight prior to dosing and until 3

- 4

hours after administration of the test substance. Water was available.

Frequency Sin gle dosage, on Day 1.

Dose level (volume) 2000 mg/kg (2.697 mL /kg) body weight.

Dose volume calculated as

( dose level (g/kg) / specific gravity ))/purity (70.7%)

6.6. Observations

Mortality/Viability Twice daily.

Body weights Days 1 (pre

- administration), 8 and 15

. Clinical signs At periodic intervals on the day of dosing (Day 1) and once daily

thereafter, until Day 15. The symptoms were graded according to fixed

scales and the time of onset, degree and duration were recorded:

Maximum grade 4: grading slight (1) to very severe (4)

Maximum grade 3: grading slight (1) to severe (3)

Maximum grade 1: presence is scored (1).

Necropsy At the end of the observation period, all animals were sacrificed by

oxygen/carbon dioxide procedure and subjected to necropsy

. Descriptions of all internal macroscopic abnormalities were recorded.

6.7. Electronic data capture

Observations/measurements in the study were recorded electronically using the following programs:

REES Centron Environmental Monitoring system version SQL 2.0 (REES scientific, Trenton, NJ,

USA) ; TOXDATA version 8.0 (WIL Research Europe B.V., ‘s

- Hertogenbosch, The Netherlands):

Clinical signs, Body weights.

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### 6.8. Interpretation

The results were evaluated according to :

- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United

Nations (2011 ),

- Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008

on classification, labelling and packaging of substances and mixtures

.

6.9. List of deviations

6.9.1. List of protocol deviations

1. Deviations from the maximum level of daily mean r elative humidity occurred.

Evaluation: Laboratory historical data do not indicate an effect of the deviations.

2. Inadvertently, for the first animal a dose volume of 2.679 mL/kg was used instead of 2.697

mL/kg, this had no effect on the dosed volume (0.41 mL) nor on the dose level (2000 mg/kg)

. Evaluation: The deviation had no effect on the study integrity.

The study integrity was not adversely affected by the deviations.

6.9.2. List of standard operating procedures deviations

Any deviations from standard operating procedures were evaluated and filed in the study file. There

were no deviations from standard operating procedures that affected the integrity of the study.

7. RESULTS

7.1. Mortality ( Table 1)

No mortality occurred.

7.2. Clinical s igns (Table 2)

Hunched posture and piloerection were noted among the majority of animals between Day s 1 and 3.

7.3. Body w eights (Table 3)

T he body weight gain shown by the animals over the study period was similar to that of normal

untreated ani mals of the same age and strain

.

7.4. Macroscopic f indings (Table 4)

No abnormalities were recorded at macroscopic post mortem examination of the animals.

8. CONCLUSION

The oral LD50 value of Castor Oil, Sulfated, Sodium Salt, 75% i n Wistar rats was established to

exceed 2000 mg active ingredient /kg body weight

.

Based on these results

, Castor Oil, Sulfated, Sodium Salt, 75% does not have to be classified and has

no obligatory labelling requirement for acute oral toxicity according to the:

- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United

Nations (2011 ),

- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and

mixtures

.

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### Table 1 Mortal ity Data

TEST DAY

HOURS AFTER TREATMENT 1

0 1

2 1

4 2 3 4 5 6 7 8 9 10 11 12 13 14 15 '

FEMALES 2000 MG/KG

- - - - - - - - - - - - - - - - -

.



### Table 2 Clinical Signs

TEST DAY

HOURS AFTER TREATMENT MAX

GRADE 1

0 1

2 1

4 2 3 4 5 6 7 8 9 10 11 12 13 14 15 '

FEMALES 2000 MG/KG

ANIMAL 1

. No clinical signs noted

- - - - - - - - - - - - - - - - -

.

FEMALES 2000 MG/KG

ANIMAL 2

. Posture

.

Hunched posture (1)

- 1 1 1 1

- - - - - - - - - - - -

. Skin / fur

.

Piloerection (1)

- 1 1 1

- - - - - - - - - - - - -

. ANIMAL 3

. Posture

.

Hunched posture (1)

- 1 1 1

- - - - - - - - - - - - -

. Skin / fur

.

Piloerection (1)

- 1 1 1

- - - - - - - - - - - - -

. ANIMAL 4

. Posture

.

Hunched posture (1)

- 1 1 1

- - - - - - - - - - - - -

. Skin / fur

.

Piloerection (1)

- 1 1 1

- - - - - - - - - - - - -

. ANIMAL 5

. Posture

.

Hunched posture (1)

- 1 1

- - - - - - - - - - - - - -

. Skin / fur

.

Piloerection (1)

- 1 1 1

- - - - - - - - - - - - -

.

- = Sign not observed

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### Table 3 Body Weights (gram)

SEX/DOSE LEVEL ANIMAL DAY 1 DAY 8 DAY 15 '

FEMALES 2000 MG/KG

1 154 180 201

.

. MEAN 154 180 201

. ST.DEV.

--- --- ---

. N 1 1 1

.

.

FEMALES 2000 MG/KG

2 171 194 217

. 3 187 219 240

. 4 161 192 201

. 5 171 201 217

.

. MEAN 1 73 202 219

. ST.DEV. 11 12 16

. N 4 4 4

.

.



### Table 4 Macroscopic Findings

ANIMAL ORGAN FINDING DAY OF DEATH '

FEMALES 2000 MG/KG

1 No findings noted Scheduled necropsy

. Day 15 after treatment

.

FEMALES 2000 MG/KG

2 No findings noted Scheduled necropsy

. Day 15 after treatment

. 3 No findings noted Scheduled necropsy

. Day 15 after treatment

. 4 No findings noted Scheduled necropsy

. Day 15 after treatment

. 5 No f indings noted Scheduled necropsy

. Day 15 after treatment

.

Note: The result of the macroscopic examination of animals 2, 3, 4, 5 was not clearly recorded in the raw data. This was

evaluated by the study director and it was considered that there was sufficient evidence to conclude that there were no findings

at necropsy.

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- APPENDIX 1 : TEST SUBSTANCE CERTIFICATE OF ANALY SIS

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