## FINAL REPORT

Study Title



## EVALUATION OF THE MU TAGENIC ACTIVITY OF

CASTOR OIL, SULFATED , SODIUM SALT, 75% IN THE

**SALMONELLA TYPHIMURI UM**  REVERSE MUTATION ASS AY

AND THE **ESCHERICHIA COLI** REVERSE MUTATION ASS AY

(WITH INDEPENDENT RE PEAT)

Author

C.M. Ver speek

- Rip

Test Facility

WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Laboratory Project Identification

Project 503340

Substance 205003/A

- Page 1 of 34

-Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 2 - 1. CONTENTS

1. CONTENTS

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

2. STATEMENT OF GLP COM PLIANCE

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

3. QUALITY ASSURANCE STATEMENT

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

4. SUMMARY

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

5. INTRODUCTION

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

5.1. Prefac e

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

5.2. Aims of the study

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

5.3. Guidelines

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

5.4. Storage and retention of records and materials

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

6. MATERIALS AND METHOD S

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

6.1. Test substance

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

6.1.1. Test substance information

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

6.1.2. Study specific test substance information

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

6.1.3. Test substance preparation

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

6.2. Reference substances

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

6.2.1. Negative control

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

6.2.2. Positive controls

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

6.3. Test system

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

6.4. Cell culture

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

6.5. Metabolic activation system

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

6.5.1. Preparation of S9

- fraction

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

6.5.2. Preparation of S9

- mix

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

6.6. Study design

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

6.6.1. Dose range finding test

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

6.6.2. Mutation as say

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

6.6.3. Colony counting

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

6.7. Electronic data capture

........................................................................................................... 13

6.8. Interpretation

........................................................................................................................... 13

6.8.1. Acceptability of the assay

............................................................................................. 13

6.8.2. Data evaluation and statistical procedures

................................................................... 13

6.9. List of deviations

..................................................................................................................... 13

6.9.1. List of protocol deviations

............................................................................................. 13

6.9.2. List o f standard operating procedures deviations

......................................................... 14

7. RESULTS

................................................................................................ ....................................... 14

7.1. Dose range finding test/first mutation experiment

................................................................... 14

7.2. Second mutation experiment

.................................................................................................. 15

8. DISCUSSION AND CONCL USION

................................................................................................ 16

9. REFE RENCES

............................................................................................................................... 16

TABLES

Table 1 Toxicity of Castor Oil, Sulfated, Sodium Salt, 75%in the dose range finding

test/first mutation experiment

........................................................................................ 15

Table 2 Toxicity of Castor Oil, Sulfated, Sodium Salt, 75%in the second mutation

experiment

.................................................................................................................... 16

Table 3 Dose range finding test: Mutagenic response of Castor Oil, Sulfated, Sodium Salt,

75% in the Salmonella typhimurium reverse mutation assay and in the

Escherichia coli reverse mutation assay

...................................................................... 17

Table 4 Experiment 1: Mutagenic response of Castor Oil, Sulfated, Sodium Salt, 75% in

the Salmonella typhimurium reverse mutation assay

................................................... 18

Table 5 Experiment 2: Mutagenic response of Castor Oil, Sulfated, Sodium Salt, 75% in

the Salmonella typhi murium reverse mutation assay and in the Escherichia coli

reverse mutation assay

................................................................................................ . 19Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 3 - APPENDICES

APPENDIX 1 SUMMARY TABLES

..................................................................................................... 17

APPENDIX 2 SUPPORTING MATERIALS AND METHOD

............................................................... 20

APPENDIX 3 DETAILED TABLES

...................................................................................................... 21

APPENDIX 4 HISTORICAL CONTROL DATA OF THE SOLVENT CONTROL

................................ 32

APPENDIX 5 HISTORICAL CONTROL DATA OF THE POSITIVE CONTROL SUBSTANCES

....... 33

APPENDIX 6 CERTIFIC ATE OF ANALYSIS

...................................................................................... 34Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 6 - 4. SUMMARY

Evaluation of the mutagenic activity of Castor Oil, Sulfated, Sodium Salt, 75% in the Salmonella

typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay (with independent

repeat).

Castor Oil, Sulfated, Sodium Salt, 75% was tested in the S almonella typhimurium reverse mutation

assay with four histidine

- requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and

TA100) and in the Escherichia coli reverse mutation assay with a tryptophan

- requiring strain of

Escherichia coli (WP 2 uvrA ). The test was performed in two independent experiments in the presence

and absence of S9

- mix (rat liver S9

- mix induced by a combination of phenobarbital and

ß- naphthoflavone).

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch R- 10135

- 064 of Castor Oil, Sulfated, Sodium Salt, 75% was a c lear amber liquid . At

concentrations of 10 mg/ml and higher the test substance was suspended in ethanol . At concentrations

of 3.33 mg/ml and lower the test substance was dissolved in ethanol

.

During the performance of the study, the test substance used in this experiment was handled as if it

had a purity of 70.7%. (as initially given by the sponsor) and a correction factor of 1.41 was used in

this assay so that all doses mentioned in this report are based on the purity of the test substance. In

the course of the development it has been identified that the test substance had a purity of 74.4 %.

Therefore, the actual concentrations were 5.2 % higher than stated in the report. The conclusion of this

report remains unchanged.

In the dose range finding test, Castor Oil, Sulfated, Sodium Salt, 75% was tested up to concentrations

of 5000 μg/plate in the absence and presence of S9

- mix in the strains TA100 and WP 2 uvrA. Castor Oil,

Sulfated, Sodium Salt, 75% precipitated on the plates at dose levels of 3330 and 5000 μg/plate. Toxicity

was only observed in tester strain TA100 in the absence and presence of S9

- mix at dose levels of 3330

and 5000 μg/plate

.

Based on the results of the dose range finding test, Castor Oil, Sulfated, Sodium Salt, 75% was tested

in the first mutation assay at a concentration range of 100 to 5000 μg/plate in the absence and presence

of 5% (v/v) S9

- mix in tester strains TA1535, TA1537 and TA98. The test substance did not precipitate

on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background

lawn , was observed in the tester strain s TA153 5 and T A 1537 in the absence of S9

- mix at the highest

tested concentration and a biologically relevant decrease in the number of revertant colonies in tester

strain TA1 537 in the pre sence of S9

- mix at the highest tested concentration

.

In an independent repeat of the assay with additional parameters, Castor Oil, Sulfated, Sodium Salt,

75% was tested at the same concentration range as the first assay in the absence and pr esence of

10% (v/v) S9

- mix in tester strains TA1535, TA1537, TA98, TA100 and WP 2 uvrA. No or a slight

precipitate of Castor Oil, Sulfated, Sodium Salt, 75% was observed at the top dose of 5000 μg/plate.

Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial

background lawn, was observed in all tester strain s , except in tester strain TA1535 in the presence of

S9

- mix and WP 2 uvrA in the absence and presence of S9

- mix

.

Castor Oil, Sulfated, Sodium Salt, 75% did not induce a significant dose

- related increase in the number

of revertant (His + ) colonies in any of the four tester strains (TA1535, TA1537, TA98 and TA100) or in the

number of revertant (Trp + ) colonies in tester strain WP 2 uvrA both in the absence and presence of S9

- metabolic activation. These results were confirmed in an independently repeated experiment.

The negative control values were within the laboratory historical control data ranges

, except for TA1535

in the first experiment. However, since these values were just outside the limit of the range, the validity

of the test was considered to be not affected. The strain

- specific positive control values were at least

three times the concurrent vehicle control group mean indicating that the test conditions were adequate.Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 7 - Based on the results of this study it is concluded that Castor Oil, Sulfated, Sodium Salt, 75% is not

mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse

mutation assay.Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 8 - 5. INTRODUCTION

5.1. Preface

Sponsor Sulfated Castor Oil Consortium, Society of Chemical

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 277 05

USA

Test Facility WIL Research Europe B.V.

Hambakenwetering 7

5231 DD ‘s

- Hertogenbosch

The Netherlands

Study Director C.M. Verspeek

- Rip

ineke.verspeek@wilresearch.com

Study Plan Start : 14 October 2013

Completion : 21 November 2013

5.2. Aims of the study

The objective of this study was to evaluate the test substance for its ability to induce reverse mutations

in a gene of histidine

- requiring Salmonella typhimurium bacterial strains resulting in histidine

independent strains, and in a gene of tryptophan

- requiring Escherichia coli bacterial strain resulting in

a tryptophan

- independent strain.

Background of the test system

The Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay

have been shown to be rapid and adequate indicators for the mutagenic activity of a wide range of

chemical compounds.

The assay was conducted in the absence and presence of a metabolizing system (S9

- mix).

The Salmonella typhi murium strains used in this study were TA1535, TA1537, TA98 and TA100. The

Escherichia coli strain used was WP 2 uvrA. The strains TA1537 and TA98 are capable of detecting

frameshift mutagens, strains TA1535, TA100 and WP 2 uvrA are capable of detecting base -p air

substitution mutagens (1

- 5).

5.3. Guidelines

The study procedures described in this report were based on the following guidelines:

- Organisation for Economic Co

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

of Chemicals; Guideline no. 471: "Genetic Toxicology: Bacterial Reverse Mutation Test" (Adopted

July 21, 1997).

- European Community (EC). Co mmission regulation (EC) No. 440/2008, Part B: Methods for the

Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: R everse

Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 3 1 May 2008.Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 9 - 5.4. Storage and retention of records and materials

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 after finalization of the report. After this period the sample will be destroyed.

6. MATERIALS AND METHOD S

6.1. Test substa nce

6.1.1. Test substance information

Identification Castor Oil, Sulfated, Sodium Salt, 75%

CAS Number 68187

- 76

- 8

Description Clear amber liquid

Batch R

- 10135

- 064

Purity/Composition 74.4 %

Test substance storage At room temperature in the dark

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

See APPENDIX 6 ; Certificate of analysis.

6.1.2. Study specific test substance information

Purity/composition correction factor

required Yes, correction factor is 1. 34 ( 74.4 % purity )

Specific Gravity / Density 1.049

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

maximum duration: until dry

Stability in vehicle Ethanol : Not indicated

Solubility in vehicle Ethanol : Partial

6.1.3. Test substance preparation

The purity of Castor Oil, Sulfated, Sodium Salt, 75% originally determined by the Sponsor was 70.7%.

All concentrations were therefore corrected by using a correction factor of 1.41, so that all doses

mentioned in this report are based on the purity of the test subs tance. In the course of the

development it has been identified that the test substance had a purity of 74.4 %. Therefore, the actual

concentrations were 5.2 % higher than stated in the report. The conclusion of this report remains

unchanged.

A solubility test was performed in project 503341. The test substance could not be dissolved in water

or in dimethyl sulfoxide since no workable suspensions were obtained. Since the test substance

formed in ethanol a workable suspension, ethanol was selected as solvent. At concentrations of

10 mg/ml and higher Castor Oil, Sulfated, Sodium Salt, 75% formed a hazy suspension in ethanol

(Merck, Darmstadt, Germany). At concentrations of 3.33 mg/ml and lower the test substance was

dissolved in ethanol.

Test substance concentr ations were used within 2 hours after preparation.Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 10

- 6.2. Reference substances

6.2.1. Negative control

The vehicle of the test substance

, which was ethanol

.

6.2.2. Positive controls

Without metabolic activation (

- S9

- mix):

Strain Chemical Concentration/plate Solvent

TA1535 so dium azide (SA) 5 μg Saline

( Sigma Aldrich Chemie, Steinheim, Germ any )

TA1537 ICR

- 191 (Acros Organics, Geel, Belgium) 2.5 μg DMSO

TA98 2- nitrofluorene (NF) (Merck) 10 μg DMSO

TA100 methylmethanesulfonate (MMS) (Sigma) 650 μg DMSO

WP 2 uvrA 4- nitroquino line N

- oxide (4

- NQO) (Sigma) 10 μg DMSO

With metabolic activation (+S9

- mix):

The positive control substance used for all tester strains was 2

- aminoanthracene (2AA) (Sigma). The

following concentrations were used:

Strain Concentration/plate Amount of S9 -m ix Solvent

TA1535 2.5 μg 5 and 10% DMSO

TA1537 2.5 μg 5% DMSO

TA1537 5 μg 10% DMSO

TA98 1 μg 5 and 10% DMSO

TA100 1 μg 5% DMSO

TA100 2.5 μg 10% DMSO

WP 2 uvrA 15 μg 5 and 10% DMSO

Solvents for reference substances

Saline = physiological saline ( Eurovet Animal Health, Bladel, The Netherlands )

DMSO = dimethyl sulfoxide ( SeccoSolv, Merck, Darmstadt, Germany )

6.3. Test system

Test system Salmonella typhimurium bacteria and Escherichia coli bacteria

Rationale Recommended test system in international guidelines (e.g. OECD, EC).

Source Trinova Biochem GmbH, Germany (Master culture from Dr. Bruce N. Ames)

(TA1535: 2006, TA1537: 2009, TA98: 2006, TA100: 2006) and (Master culture

from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK)

(WP 2 uvrA, 2008)

The characteristics of the different Salmonella typhimurium strains were as follows:

Strain Histidine mutation Mutation type

TA1537 his C3076 Frameshift

TA98 his D3052/R

- factor* Frameshift

TA1535 his G46 Base

- pair substit utions

TA100 his G46/R

- factor* Base

- pair substitutions

*: R

- factor = plasmid pKM101 (increases error

- prone DNA repair)

Each tester strain contained the following additional mutations:

rfa : deep rough (defective lipopolysaccharide cellcoat)

gal : mutation in the galactose metabolism

chl : mutation in nitrate reductase

bio : defective biotin synthesis

uvrB : loss of the excision repair system (deletion of the ultraviolet

- repair B gene)Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 11

- The Salmonella typhimurium strains a re regularly checked to confirm their histidine

- requirement,

crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV

- sensitivity and the number of

spontaneous revertants.

The Escherichia coli WP

2 uvrA strain detects base

- pair substitutions. The strain lacks an excision repair

s ystem and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of

mutagens has been enhanced by permeabilization of the strain using Tris

- EDTA treatment (Ref.1).

The strain i s regularly checked to confirm the tryptophan

- requir ement, UV

- sensitivity and the number

of spontaneous revertants.

Stock cultures of the five strains were stored in liquid nitrogen (

- 196°C).

6.4. Cell culture

Preparation of bacterial cultures

Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD,

Hampshire, England) and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached

an optical density of 1.0 ± 0. 1 at 700 nm (10 9 cells/ml). Freshly grown cultures of each strain were

used for a test.

Agar plates

Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter:

18 g purified agar ( Merck ) in Vogel

- Bonner Medium E, 20 g glucose (Fres e nius Kabi, Bad Homburg,

Germany ). The agar plates for the test with the S almonella typhimurium strains also contained

12.5 μg/plate biotin ( Merck) and 15 μg/plate histidine ( Merck) and the agar plates for the test with the

Escherichia coli strain contained 15 μg/plate tryptophan (Acros Organics).

Top agar

Milli

- Q water containing 0.6% (w/v) bacteriological agar ( Merck ) and 0.5% (w/v) sodium chloride

( Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass

tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

Environmental conditions

All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual

range 36.4

– 37.7°C) in the dark.

6.5. Metabolic activation system

R at liver microsomal enzymes (S9 homogenate) were obtained from Tr inova Biochem GmbH,

Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally

with a suspension of phenobarbital (80 mg/kg body weight) and ß

- naphthoflavone (100 mg/kg), except

in the second experiment in tester strain WP 2 uvrA

. In this part of the experiment , r at liver microsomal

enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles

River, Sulzfeld, Germany (see protocol deviation 1)

.

6.5.1. Preparation of S9

- fraction

The animals were house d at WIL Research Europe in a special room under standard laboratory

conditions, as described in the Standard Operating Procedures. The rats were orally dosed for three

consecutive days with a suspension of phenobarbital (Bufa B.V., IJsselstein, The Nether lands )

(80 mg/kg body weight) and ß

- naphthoflavone (Sigma Aldrich Chemie) (100 mg/kg body weight) in

corn oil (Fagron Farmaceutical, Capelle aan de IJssel, The Netherlands ) (they were denied access to

food for 3 to 4 hours preceding each dosing). One day after the final exposure (24 h), the rats were

sedated using oxygen/carbon dioxide and then killed by decapitation. The rats received a limited

quantity of food during the night before sacrifice. The livers of the rats were removed aseptically, and

washed i n cold (0°C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na

2 - EDTA

(Merck). Subsequently the livers were minced in a blender and homogenized in 3 volumes ofCastor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 12

- phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g.

The supernatant (S9) was transferred into sterile ampoules, which were stored in liquid nitrogen

(- 196°C) for a maximum of 1 year.

Before use, all S9 batches were characterised with the mutagens Benzo

- (a)

- pyrene (Sigma) and

2- aminoanth racene (Sigma), which require metabolic activation, in tester strain TA98 at concentrations

of 5 μg/plate and 1 μg/plate, respectively.

6.5.2. Preparation of S9

- mix

S9

- mix was prepared immediately before use and kept on ice. S9

- mix contained per 10 ml: 30 mg

NAD P ( Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose -6- phosphate

(Roche Diagnostics, Mannheim, Germany) in 5.5 ml or 5.0 ml Milli

- Q water (first or second experiment

respectively) (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml

0.08 M MgCl

2 solution (Merck) ; 1 ml 0.33 M KCl solution (Merck) . The above solution was filter

(0.22 μm)

- sterilized. To 9.5 ml of S9

- mix components 0 .5 ml S9

- fraction was added (5% (v/v) S9

- fraction) to complete the S9

- mix i n the first experiment and to 9.0 ml of S9

- mix components 1.0 ml

S9

- fraction was added (10% (v/v) S9

- fraction) to complete the S9

- mix in the second experi ment.

6.6. Study design

6.6.1. Dose range finding test

Selection of an adequate range of doses was based on a dose range finding test with the strains

TA100 and WP 2 uvrA, both with and without 5% (v/v) S9

- mix. Eight concentrations, 3, 10, 33, 100, 333,

1000, 3330 and 5000 μg/plate were tested in triplicate. The highest concentration of Castor Oil,

Sulfated, Sodium Salt, 75% used in the subsequent mutation assay was 5 000 μ g/plate.

6.6.2. Mutation assay

At least five different doses (increasing with approximately half

- log steps) of the test substance were

tested in triplicate in each strain. In the first experiment Castor Oil, Sulfated, Sodium Salt, 75% was

tested both in the absence and presence of 5% (v/v) S9

- mix in tester strains TA1535, TA1537 and

TA98. In an independent repeat of the assay with additional parameters, the test substance was

tested both in the absence and presence of 10% (v/v) S9

- mix in all tester strains.

The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in

the presence and absence of S9

- mix.

Top agar in top agar tubes was melted by heating to 45°C. The following solutions were successively

added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (10 9 cells/ml) of one of the tester

strains, 0.1 ml of a dilution of the test substance in ethanol and either 0.5 ml S9

- mix (in case of

activation ass ays) or 0.5 ml 0.1 M phosphate buffer (in case of non

- activation assays). The ingredients

were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate.

After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C

for 48 ± 4 h. After this period revertant colonies histidine independent (His + ) for Salmonella

typhimurium bacteria and tryptophan independent (Trp + ) for Escherichia coli were counted.

6.6.3. Colony counting

The revertant colonies wer e counted automatically with the Sorcerer Colony Counter. Plates with

sufficient test article precipitate to interfere with automated colony counting were counted manually

and evidence of test article precipitate on the plates was recorded. The condition of the bacterial

background lawn was evaluated, both macroscopically and microscopically by using a dissecting

microscope.Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 13

- 6.7. Electronic data capture

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

programme:

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

USA).

- Ames study Manager version 1.23 (Perceptive Instruments Ltd.)

6.8. Interpretation

6.8.1. Acceptability of the assay

A Salmonella typhimurium reverse mutation assay a nd/or Escherichia coli reverse mutation assay is

considered acceptable if it meets the following criteria:

a) The negative control data (number of spontaneous revertants per plate) should be within the

laboratory historical range for each tester strain (see APPENDIX 4 )

. b) The positive control chemicals should produce responses in all tester strains, which are within the

laboratory historical range documented for each positive control substance. Furthermore, the mean

plate count should be at least three times the concurrent vehicle control group mean (see

APPENDIX 5 )

. c) The selected dose range should include a clearly toxic concentration or should exhibit limited

solubility as demonstrated by the preliminary toxicity range

- finding test or should extend to

5 mg/plate.

6.8.2. Data evaluation and statistical procedures

No formal hypothesis testing was done.

A t est substance is considered negative (not mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 is not greater than two (2) times the

concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or

WP 2 uvrA is not greater than three (3) times the concurrent vehicle control.

b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:

a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent

control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP 2 uvrA is

greater than three (3) times the concurrent vehicle control.

b) In case a repeat experiment is performed when a positive response is observed in one of the tester

strains, the positive response should be reproducible in at least one independently repeated

experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final

evaluation decision.

6.9. List of deviations

6.9.1. List of protocol deviations

1. In the mutatio n experiment (except in experiment 2, WP 2 uvrA), t he S9

- fraction was obtained from

Trinova Biochem

. Evaluation: Due to a shortage of the S9 homogenate prepared at WIL Research Europe, S9

homogenate obtained from Trinova Biochem was used

. The use of another S9 homogenate had

no effect on the results of the study.

2. In the first experiment, the mean plate counts of the solvent control of TA1535 in the absence and

presence of S9

- mix were not within the laboratory historical range

. Evaluation: Mean plate counts of 26 and 31 revertants (in the absence and presence of S9

- mix

, respectively) were just above the limit of the range (25)

, therefore this deviation in the mean plate

count of the solvent controls had no effect on the results of the study.Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 14

- 3. Fifteen μg/plate 2- amino anthracene was used as positive control substance in the presence of

S9

- mix for tester strain WP 2 uvrA

. Evaluation: To be sure that a clear positive response was obtained, the concentration of the

positive control substance was increased. This had no influence on the study integrity.

4. The positive control substances of the following tester strains: tester strain TA1535 (absence of

S9

- mix, first and second experiment), TA100 (presence of S9

- mix , second experiment ) and

WP 2 uvrA ( absence of S9

- mix, dose ra nge f inding test and second experiment) showed

responses (mean plate count) which were not within the laboratory historical range.

Evaluation: The values were above the limit of the range. These values were more than 3 times

greater than the concurrent solvent control values, therefore this deviation in the mean plate

count of the positive controls had no effect on the results of the study.

5. In the dose range finding test, the mean plate count of the positive control of WP 2 uvrA in the

presence of S9

- mix was not within the laboratory historical range

. Evaluation: Although the mean value of 99 was below the limit of the range (122), the mean value

was more than 3 times greater than the concurrent solvent control value, therefore this deviation

in the mean plate count of the positive control had no effect on the results of the study.

6. The experimental end date was 21 November 2013, which is not within the period mentioned in

the time schedule in the protocol.

Evaluation: This deviation in the experimental end date had no effect on the results of the study.

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. Dose range finding test/first mutation experiment

Castor Oil, Sulfated, Sodium Salt, 75% was tested in the tester strains TA100 and WP 2 uvrA wit h

concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 μg/plate in the absence and presence of

S9

- mix. Based on the results of the dose range finding test, the following dose range was selected for

the first mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and

presence of S9

- mix: 100, 333, 1000, 3330 and 5000 μg/plate.

The results are shown in Table 3 and Table 4

. The individual data are presented in APPENDIX 3

.

Precipitate

I n the dose range finding test

, p recipitation of Castor Oil, Sulfated, Sodium Salt, 75% on the plates

was observed at the start and at the end of the incubation period at concentrations of 3330 and

5000 μg/plate. In the first mutation experiment, precipitation of Castor Oil, Sulfated, Sodium Salt, 75%

on the plates was observed at the start of the incubation period at the concentration of 5000 μg/plate

and no precipitation on the plates was observed at the end of the incubati on period

.

Toxicity

To determine the toxicity of Castor Oil, Sulfated, Sodium Salt, 75%, the reduction of the bacterial

background lawn, the increase in the size of the microcolonies and the reduction of the revertant

colonies were examined. The definitio ns are stated in APPENDIX 2

.

In tester strain TA98 and WP 2 uvrA, no reduction of the bacterial background lawn and no biologically

relevant decrease in the number of revertants were observed.Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 15

- The reduction of the bacterial background lawn and the reduction in the number of revertant in the

other tester strains are presented in Table 1

.

Table 1 Toxicity of Castor Oil, Sulfated, Sodium Salt, 75%in the dose range finding

test/first mutation experiment

(Reduction of the bacterial background lawn and in the number of revertant colonies)

Strain Without S9

- mix With S9

- mix

Dose Bacterial Revertant

(μg/plate) background lawn colonies Dose Bacterial Revertant

(μg/plate) background lawn colonies

TA100 3330 moderate moderate

5000 moderate extreme 3330 moderate moderate

5000 moderate moderate

TA1535 5000 sligh t

- 1 5000 normal

- 1

TA1537 5000 slight

- 1 5000 normal complete

- 1 No reduction in the number of revertant colonies less than the minimal value of the historical

control data range.

Mutagenicity

N o increase in the number of revertants was observed upon treatment with Castor Oil, Sulfated,

Sodium Salt, 75% under all conditions tested.

7.2. Second mutation e xperiment

To obtain more information about the possible mutagenicity of Castor Oil, Sulfated, Sodium Salt, 75%

, a second mutation experiment was performed in the absence of S9

- mix and in the presence of

10% (v/v) S9

- mix. Based on the results of the first mutation assay, Castor Oil, Sulfated, Sodium Salt,

75% was tested up to the dose level of 5000 μg/plate in strains TA1535, TA1537, TA98, TA100 and

WP 2 uvr A. The results are shown in Table 5

, the individual data are presented in APPENDIX 3

.

Precipitate

Precipitation of Castor Oil, Sulfated, Sodium Salt, 75% on the plates was observed at the start of the

incubation period at concentration s of 3330 and 5000 μg/plate in all tester strains and at the end of the

incubation period at 5000 μg/plate in the tester strains TA1535 and TA1537 (absence of S9

- mix) and

TA100 (absence and presence and presence of S9

- mix)

.

Toxicity

In tester strain WP 2 uvrA, no reduction of the bacterial background lawn and no biologically relevant

decrease in the number of revertants were observed.Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 16

- The reduction of the bacterial background lawn and the reduction in the number of revertant in the

other tester strains are presented in Table 2

.

Table 2 Toxicity of Castor Oil, Sulfated, Sodium Salt, 75%in the second mutation

experiment

(Reduction of the bacterial background lawn and in the number of revertant colonies)

Strain Without S9

- mix With S9

- mix

Dose Bacterial Revertant

(μg/plate) background lawn colonies Dose Bacterial Revertant

(μg/plate) background lawn colonies

TA1535 3330 slight

- 1

5000 moderate

- 1 5000 normal

- 1

TA1537 3330 slight

- 1

5000 moderate extreme 5000 normal extreme

TA98 5000 slight

- 1 5000 slight

- 1

TA100 3330 moderate moderate

5000 moderate extreme 3330 slight

- 1

5000 moderate moderate

- 1 No reduction in the number of revertant colonies less than the minimal value of the historical

control data range.

Muta genicity

In second mutation assay, no increase in the number of revertants was observed upon treatment with

Castor Oil, Sulfated, Sodium Salt, 75% under all conditions tested.

8. DISCUSSION AND CONCL USION

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose

related increase in the number of revertants in two independently repeated experiments.

The negative control values were within the laboratory historical control data ranges, except the

response for TA153 5 in the absence of S9

- mix, first experiment (see protocol deviation 2 ).

The strain

- specific positive control values were within the laboratory historical control data ranges

indicating that the test conditions were adequate and that the metabolic activat ion system functioned

properly, except the response s for TA1535, TA100 and WP 2 uvrA ( see protocol deviation s 4 and 5 ).

Based on the results of this study it is concluded that Castor Oil, Sulfated, Sodium Salt, 75% is not

mutagenic in the Salmonella typhimu rium reverse mutation assay and in the Escherichia coli reverse

mutation assay.

9. REFERENCES

1 Leonardo, J.M., Dornfeld, S.S. and Peak, M.J., 1984, Evaluation of E. coli K12 343 \ 113 and

derived strains for microbial mutagenicity assays. Mutation Res., 130

, 87

- 95.

2 Ames, B.N., McCann, J. and Yamasaki, E., 1975, Methods for detecting carcinogens and

mutagens with the Salmonella/ mammalian microsome mutagenicity test, Mutation Res., 31 , 347

-

364.

3 Maron, D.M. and Ames, B.N., 1983, Revised methods for the Salmonell a mutagenicity test,

Mutation Res., 113 , 173

- 215. Erratum, 1983, Mutation Res., 113 , 533.

4 Green, M.H.L. and Muriel, W.J., 1976, Mutagen testing using Trp + reversion in Escherichia coli

, Mutation Res., 38 , 3

- 32.

5 Vogel, H.J. and Bonner, D.M., 1956, Acetylornithinase of Escherichia coli : partial purification and

some properties. J. Biol. Chem., 218 , 97

- 106.Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 17

- APPENDIX 1 SUMMARY TABLES

Table 3 Dose range finding test: Mutagenic response of Castor Oil, Sulfated, Sodium

Salt, 75% in the **Salmonella typhimurium** reverse mutation assay and in the

**Escherichia coli** reverse mutation assay

Dose

(μg/plate) Mean number of revertant colonies/3 replicate plates ( ± S.D.) with one Salmonella

typhimurium and one Escherichia coli strain

.

TA100 WP2uvrA

Without S9

- mix

Positive control 927 ± 85 1499 ± 97

Solvent control 136 ± 24 n NP 34 ± 10 n NP

3 126 ± 18 28 ± 2

10 130 ± 15 24 ± 6

33 123 ± 16 21 ± 3

100 111 ± 9 26 ± 10

333 102 ± 12 25 ± 3

1000 101 ± 4 n NP 32 ± 8 NP

3330 41 ± 4 m SP 10 ± 5 SP

5000 24 ± 4 m SP 17 ± 3 n SP

With S9

- mix 1

Positive control 1670 ± 710 99 ± 11

Solvent control 99 ± 30 n NP 31 ± 8 n NP

3 116 ± 4 26 ± 8

10 106 ± 10 23 ± 6

33 140 ± 1 31 ± 4

100 120 ± 4 32 ± 8

333 110 ± 7 35 ± 3

1000 90 ± 3 n NP 22 ± 8 NP

3330 51 ± 9 m SP 16 ± 2 SP

5000 41 ± 8 m SP 19 ± 5 n SP

1 Plate incorporation assay (5% S9)

NP No precipitate

SP Slight Precipitate

m Bacterial background lawn moderately reduced

n Normal bacterial background lawnCastor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 18

- APPENDIX 1 

## – continued 

## –

Table 4 E xperiment 1 : Mutagenic response of Castor Oil, Sulfated, Sodium Salt , 75% in

the **Salmonella typhimurium** reverse mutation assay

Dose

(μg/plate) Mean number of revertant colonies/3 replicate plates ( ± S.D.) with

different strains of Salmonella typhimurium

.

TA1535 TA1537 TA98

Without S9

- mix

Positive control 15 49 ± 56 753 ± 95 942 ± 19

Solvent control 26 ± 2 n NP 7 ± 5 n NP 25 ± 10 n NP

100 18 ± 1 6 ± 2 14 ± 5

333 25 ± 4 6 ± 1 26 ± 4

1000 23 ± 3 6 ± 4 18 ± 4

3330 10 ± 3 n 4 ± 3 n 22 ± 0

5000 7 ± 3 s NP 4 ± 1 s NP 19 ± 4 n NP

Wi th S9

- mix 1

Positive control 257 ± 32 385 ± 11 1135 ± 77

Solvent control 31 ± 4 n NP 10 ± 2 n NP 31 ± 13 n NP

100 24 ± 8 9 ± 2 33 ± 13

333 26 ± 7 9 ± 2 28 ± 7

1000 20 ± 1 6 ± 2 24 ± 4

3330 18 ± 4 3 ± 3 20 ± 8

5000 13 ± 2 n NP 0 ± 1 n N P 23 ± 4 n NP

1 Plate incorporation assay (5% S9)

NP No precipitate

n Normal bacterial background lawn

s Bacterial background lawn slightly reducedCastor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 19

- APPENDIX 1 

## – continued 

## –

Table 5 Experiment 2: Mutagenic resp onse of Castor Oil, Sulfated, Sodium Salt, 75% in

the **Salmonella typhimurium** reverse mutation assay and in the **Escherichia coli**

reverse mutation assay

Dose

(μg/plate) Mean number of revertant colonies/3 replicate plates ( ± S.D.) with

different strains of Salmonella typhimurium and one Escherichia coli strain.

TA1535 TA1537 TA98 TA100 WP2uvrA

Without S9

- mix

Positive control 1670 ± 57 507 ± 34 621 ± 24 1062 ± 69 1485 ± 57

Solvent control 21 ± 3 n NP 10 ± 4 n NP 25 ± 5 n NP 90 ± 14 n NP 4 9 ± 8 n

100 18 ± 1 6 ± 3 19 ± 6 90 ± 2 47 ± 9

333 18 ± 3 5 ± 2 22 ± 4 92 ± 12 40 ± 9

1000 16 ± 2 NP 7 ± 2 n 17 ± 7 69 ± 8 n 45 ± 9

3330 7 ± 1 s NP 2 ± 3 s NP 16 ± 4 n 40 ± 13 m NP 19 ± 5

5000 3 ± 4 m SP 0 ± 1 m SP 13 ± 3 s NP 24 ± 2 m SP 25 ± 8 n NP

With S9

- mix 1

Positive control 204 ± 14 392 ± 179 1197 ± 58 1949 ± 97 277 ± 20

Solvent control 22 ± 6 n NP 7 ± 4 n NP 27 ± 3 n NP 79 ± 4 n NP 47 ± 5 n

100 20 ± 5 11 ± 1 29 ± 5 69 ± 3 52 ± 11

333 24 ± 5 6 ± 2 26 ± 5 89 ± 15 5 4 ± 9

1000 17 ± 8 8 ± 4 30 ± 7 81 ± 12 n 39 ± 10

3330 12 ± 3 2 ± 1 24 ± 1 n 62 ± 4 s NP 32 ± 9

5000 7 ± 3 n NP 0 ± 1 n NP 15 ± 5 s NP 39 ± 8 m SP 30 ± 7 n NP

1 Plate incorporation assay (10% S9)

NP No precipitate

SP Slight Precipitate

m Ba cterial background lawn moderately reduced

n Normal bacterial background lawn

s Bacterial background lawn slightly reducedCastor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 20

- APPENDIX 2 SUPPORTING MATERIALS AND METHOD

Bacterial background lawn evaluation

The condition of the bacterial background lawn is evaluated (if indicated), both macroscopically and

microscopically by using a dissecting microscope (results are normal unless indicated in tables).

Definition Characteristics

Normal Distinguished by a healthy microcolony l awn.

Slightly reduced Distinguished by a slight thinning of the microcolony lawn.

Moderately reduced Distinguished by a moderate thinning of the microcolony lawn.

Extremely reduced Distinguished by an extreme thinning of the microcolony lawn and an

increas e in the size of the microcolonies compared to the solvent control

plate.

Absent Distinguished by a complete lack of any microcolony background lawn.

Precipitation evaluation

Evidence of test article precipitate on the plates is recorded by addition of the following precipitation

definition.

Definition Characteristics

Slight Precipitate Distinguished by noticeable precipitate on the plate.

However, the precipitate does not influence automated counting of the plate.

Moderate Precipitate Distinguished by a marked amount of precipitate on the plate, requiring the

plate to be hand counted.

Heavy Precipitate Distinguished by a large amount of precipitate on the plate, making the

required hand count difficult.

Evaluation of the reduction in the number of revertants

The reduction in the number of revertant colonies compared to number of revertants in the solvent

control is evaluated as follows:

A reduction of 21

- 40%: slight reduction.

A reduction of 41

- 60%: moderate reduction.

A reduction of 61

- 99%: extreme reduction.

If the size of the microcolonies was increased to small colonies due to an extremely reduced

background lawn the reduction is evaluated as microcolonies. If no revertant colonies are observed on

the plates the reduction is evaluated as a complete lack of revertants.

However, any mean plate count equal to the minimal value of the historical control data range should

be considered not toxic.Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 21

- APPENDIX 3 DETAILED TABLES

Individual plate counts; (following pages)

LIST OF ABBREVIATIONS

n Normal bacterial background lawn

s Bacterial background lawn slightly reduced

m Bacterial background lawn moderately reduced

a Bacterial background lawn absent

e Bacterial background lawn extremely reduced

i One plate infected: Mean o f two plates

MC Microcolonies

NP No precipitate

SP Slight precipitate

MP Moderate precipitate

HP Heavy precipitateCastor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 22

- APPENDIX 3 

## – continued 

## –

Dose range finding

Strain TA100

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 847 1016 917 927 ± 85

solvent control 159 n NP 112 n NP 136 n NP 136 ± 24

3 125 109 144 126 ± 18

10 147 125 118 130 ± 15

33 141 110 117 123 ± 16

100 121 106 105 111 ± 9

333 97 94 116 102 ± 12

1000 99 n NP 106 n NP 99 n NP 101 ± 4

3330 41 m SP 37 m SP 45 m SP 41 ± 4

5000 20 m SP 27 m SP 26 m SP 24 ± 4

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 853 2020 2137 1670 ± 710

solvent control 87 n NP 78 n NP 133 n NP 99 ± 30

3 112 120 116 116 ± 4

10 103 98 117 106 ± 10

33 141 140 139 140 ± 1

100 116 120 124 120 ± 4

333 106 118 107 110 ± 7

1000 90 n NP 93 n NP 88 n NP 90 ± 3

3330 60 m SP 42 m SP 52 m SP 51 ± 9

5000 35 m SP 50 m SP 37 m SP 41 ± 8Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 23

- APPENDIX 3 

## – continued 

## –

Dose range finding

Strain WP2uvrA

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1605 1415 1477 1499 ± 97

sol vent control 44 n NP 24 n NP 33 n NP 34 ± 10

3 31 27 27 28 ± 2

10 18 24 30 24 ± 6

33 19 20 24 21 ± 3

100 18 37 22 26 ± 10

333 27 26 22 25 ± 3

1000 23 NP 33 NP 39 NP 32 ± 8

3330 5 SP 10 SP 14 SP 10 ± 5

5000 19 n SP 18 n SP 14 n SP 17 ± 3

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 110 99 88 99 ± 11

solvent control 33 n NP 22 n NP 38 n NP 31 ± 8

3 34 19 26 26 ± 8

10 16 27 27 23 ± 6

33 35 27 31 31 ± 4

100 27 27 41 32 ± 8

333 34 33 39 35 ± 3

1000 20 NP 30 NP 15 NP 22 ± 8

3330 14 SP 18 SP 16 SP 16 ± 2

5000 14 n SP 20 n SP 24 n SP 19 ± 5Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 24

- APPENDIX 3 

## – continued 

## –

Experiment 1

Strain TA1535

WITHOUT S9

- MIX

plate 1 2 3 ME AN SD

dose (μg/plate)

positive control 1609 1498 1541 1549 ± 56

solvent control 27 n NP 23 n NP 27 n NP 26 ± 2

100 19 18 18 18 ± 1

333 23 29 22 25 ± 4

1000 20 26 24 23 ± 3

3330 12 n 7 n 12 n 10 ± 3

5000 1 0 s NP 4 s NP 8 s NP 7 ± 3

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 230 248 292 257 ± 32

solvent control 26 n NP 33 n NP 34 n NP 31 ± 4

100 16 31 24 24 ± 8

333 33 26 2 0 26 ± 7

1000 20 20 21 20 ± 1

3330 18 22 15 18 ± 4

5000 15 n NP 11 n NP 12 n NP 13 ± 2Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 25

- APPENDIX 3 

## – continued 

## –

Experiment 1

Strain TA1537

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 850 661 747 753 ± 95

solvent control 1 n NP 8 n NP 11 n NP 7 ± 5

100 4 7 8 6 ± 2

333 5 7 7 6 ± 1

1000 1 8 8 6 ± 4

3330 3 n 7 n 1 n 4 ± 3

5000 4 s NP 4 s NP 5 s NP 4 ± 1

WITH S9

- MIX

plate 1 2 3 MEAN S D

dose (μg/plate)

positive control 381 377 397 385 ± 11

solvent control 11 n NP 7 n NP 11 n NP 10 ± 2

100 8 7 11 9 ± 2

333 8 8 12 9 ± 2

1000 4 7 8 6 ± 2

3330 7 1 1 3 ± 3

5000 0 n NP 1 n NP 0 n NP 0 ± 1Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 26

- APPENDIX 3 

## – continued 

## –

Experiment 1

Strain TA98

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 921 959 945 942 ± 19

solvent control 35 n NP 26 n NP 15 n NP 25 ± 10

100 10 12 19 14 ± 5

333 22 29 26 26 ± 4

1000 14 19 22 18 ± 4

3330 22 22 22 22 ± 0

5000 15 n NP 19 n NP 22 n NP 19 ± 4

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1223 1099 1083 1135 ± 77

solvent control 16 n NP 41 n NP 35 n NP 31 ± 13

100 20 34 46 33 ± 13

333 20 34 30 28 ± 7

1000 20 27 26 24 ± 4

3330 29 15 16 20 ± 8

5000 26 n NP 19 n NP 24 n NP 23 ± 4Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 27

- APPENDIX 3 

## – continued 

## –

Experiment 2

Strain TA1535

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1729 1666 1616 1670 ± 57

solvent control 24 n NP 20 n NP 18 n NP 21 ± 3

100 18 19 18 18 ± 1

333 20 15 20 18 ± 3

1000 1 6 NP 18 NP 15 NP 16 ± 2

3330 7 s NP 7 s NP 8 s NP 7 ± 1

5000 0 m SP 3 m SP 7 m SP 3 ± 4

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 188 215 209 204 ± 14

solvent control 15 n N P 25 n NP 26 n NP 22 ± 6

100 24 15 20 20 ± 5

333 19 29 23 24 ± 5

1000 12 26 14 17 ± 8

3330 10 15 10 12 ± 3

5000 11 n NP 5 n NP 5 n NP 7 ± 3Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 28

- APPENDIX 3 

## – continued 

## –

Experiment 2

Strain TA1537

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 472 509 540 507 ± 34

solvent control 8 n NP 15 n NP 7 n NP 10 ± 4

100 4 10 5 6 ± 3

333 3 5 7 5 ± 2

1000 8 n 4 n 8 n 7 ± 2

3330 5 s NP 0 s NP 1 s NP 2 ± 3

5000 0 m SP 0 m SP 1 m SP 0 ± 1

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 186 503 487 392 ± 179

solvent control 7 n NP 3 n NP 10 n NP 7 ± 4

100 12 10 11 11 ± 1

333 7 4 7 6 ± 2

1000 4 8 12 8 ± 4

3330 3 1 1 2 ± 1

5000 0 n NP 1 n NP 0 n NP 0 ± 1Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 29

- APPENDIX 3 

## – continued 

## –

Experiment 2

Strain TA98

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 599 646 618 621 ± 24

solvent control 29 n NP 26 n NP 19 n NP 25 ± 5

100 26 16 16 19 ± 6

333 19 22 26 22 ± 4

1000 11 15 24 17 ± 7

3330 12 n 19 n 16 n 16 ± 4

5000 14 s NP 10 s NP 16 s NP 13 ± 3

WITH S9

- MIX

plate 1 2 3 MEAN S D

dose (μg/plate)

positive control 1260 1147 1184 1197 ± 58

solvent control 23 n NP 29 n NP 29 n NP 27 ± 3

100 30 24 34 29 ± 5

333 21 31 26 26 ± 5

1000 24 29 38 30 ± 7

3330 24 n 24 n 23 n 24 ± 1

5000 15 s NP 19 s NP 10 s NP 15 ± 5Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 30

- APPENDIX 3 

## – continued 

## –

Experiment 2

Strain TA100

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1124 988 1073 1062 ± 69

solvent control 103 n NP 7 5 n NP 91 n NP 90 ± 14

100 91 91 88 90 ± 2

333 94 103 79 92 ± 12

1000 71 n 60 n 75 n 69 ± 8

3330 26 m NP 46 m NP 49 m NP 40 ± 13

5000 27 m SP 23 m SP 23 m SP 24 ± 2

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1947 1853 2047 1949 ± 97

solvent control 79 n NP 82 n NP 75 n NP 79 ± 4

100 73 67 67 69 ± 3

333 97 98 72 89 ± 15

1000 68 n 91 n 84 n 81 ± 12

3330 65 s NP 57 s NP 64 s NP 62 ± 4

5000 30 m SP 42 m SP 46 m SP 39 ± 8Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 31

- APPENDIX 3 

## – continued 

## –

Experiment 2

Strain WP2uvrA

WITHOUT S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 1551 1460 1445 1485 ± 57

solvent control 46 n 58 n 44 n 49 ± 8

100 46 38 56 47 ± 9

333 45 30 46 40 ± 9

1000 42 56 38 45 ± 9

3330 19 15 24 19 ± 5

5000 16 n NP 30 n NP 30 n NP 25 ± 8

WITH S9

- MIX

plate 1 2 3 MEAN SD

dose (μg/plate)

positive control 27 1 261 299 277 ± 20

solvent control 52 n 48 n 42 n 47 ± 5

100 48 65 44 52 ± 11

333 44 57 61 54 ± 9

1000 48 29 41 39 ± 10

3330 41 24 30 32 ± 9

5000 24 n NP 29 n NP 38 n NP 30 ± 7Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 32

- APPENDIX 4 HIST ORICAL CONTROL DATA OF THE SOLVENT CONTROL

TA1535 TA1537 TA98 TA100 WP 2 uvrA

S9

- mix

- + - + - + - + - +

Range 2

- 25 2

- 25 2

- 22 2

- 32 7

- 49 7

- 56 63

- 195 58

- 205 7

- 51 8

- 62

Mean 10 8 5 5 20 24 110 98 24 26

SD 4 3 2 3 7 7 24 24 7 7

n 1296 1319 1108 1101 1302 1304 1373 1342 1169 1155

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between July 2010 and July 2013.Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 33

- APPENDIX 5 HISTORICAL CONTROL DATA OF THE POSITIVE CONTROL SUBSTANCES

TA1535 TA1537 TA98

S9

- mix

- + - + - +

Range 250

- 1270 72

- 1016 113

- 1182 85

- 742 407

- 1518 153

- 1663

Mean 843 266 603 358 997 778

SD 130 120 243 106 147 280

n 1313 1317 1117 1118 1324 1307

TA100 WP 2 uvrA

S9

- mix

- + - +

Range 348

- 1417 229

- 1752 138

- 1479 122

- 1187

Mean 854 1113 1072 354

SD 120 238 200 126

n 1338 1332 1166 1153

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between July 2010 and July 2013.Castor Oil, Sulfated, Sodium Salt, 75% Project 503340

- Page 34

- APPENDIX 6 CERTIFICATE OF ANALYSIS