Eurofins PSL T | 732-438-5100 2394 US Highway 130 Suite E F | 732-355-3275 Dayton, NJ 08810 psl@productsafetylabs.com USA www.productsafetylabs.com 

## PRODUCT

STUDY TITLE Local Lymph Node Assay (LLNA) in Mice



## DATA REQUIREMENT U.S. EPA Health Effects Test Guidelines, OPPTS 870.2600 OECD Guidelines for the Testing of Chemicals, Test No. 429 Official Journal of the European Communities, Meth ods for the Determination of Toxicity and Other Health Effects, Part B.42 (Skin Sensitization: Local Lymph Node Assay), Commission Regulation (EC) No. 440/2008



## AUTHOR Jennifer Durando, B.S.



## STUDY COMPLETED ON August 24, 2011



## PERFORMING LABORATORY

Eurofins PSL 2394 US Highway 130 Dayton, New Jersey 08810



## SPONSOR The Dow Chemical Company Midland, MI 48674 USA



## EPSL STUDY NUMBER

DOW STUDY NUMBER

Page 1 of 41

# :::: eurofins I PSL Page 2 of 41 EPSL Study Number Dow Study Number



### STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS

No claim of confidentiality, on any basis whatsoever, is made for any information contained in this document. I acknowledge that information not designated as within the scope of FIFRA sec. 1 O( d)(l )(A), (B), or (C) and which pertains to a registered or previously registered pesticide is not entitled to confidential treatment and may be released to the public, subject to the provisions regarding disclosure to multinational entities under FIFRA sec. lO(g).

company: _______________**T=h=e~D~o~w~C=h=e=m=l=·c=al~C=o=m=?pa=n~y~----------------** __________ __

Company Agent Date: 

# g 19, Z-d/I Brandi B. McCoy Product Steward Specialist The Dow Chemical Company

THESE DATA MAY BE CONSIDERED CONFIDENTIAL IN COUNTRIES OUTSIDE THE UNITED STATES

# ~:: 

# eurofins
PSL Page 3 of 41 EPSL Study Number Dow Study Number



### GOOD LASORA TORY PRACTICE COMPLIANCE STATEMENT

This study meets the requirements of 40 CFR Part 160: U.S. EPA (FIFRA), 1989; 21 CFR Part 58: U.S. FDA, 1987; OECD (as revised in 1997) published in ENVIMCICHEM (98)17, OECD, Paris, 1998; 11- Nousan-No. 6283, 1 October, 1999: JMAFF; and EC Directive 2004110/EC, Official Journal of the European Union, L50/44, Feb. 20, 2004, with the following exceptions: the stability, uniformity of mixture and verification of concentration of the and the positive control substance, alpha-Hexylcinnamaldehyde, Technical, in their carriers, were not determined. These exceptions did not have any impact on the conduct, results, interpretation or validity of this study.

Study Director:

Applicant/Submitter:

Sponsor: Date

The Dow Chemical Company

Date

The Dow Chemical Company

DatePage 4 of 41 EPSL Study Number Dow Study Number



### QUALITY ASSURANCE STATEMENT

The Eurofins PSL Quality Assurance Unit has re view ed this final study r eport to assure the repor t accurately describes the methods and standard ope rating procedures, and that the reported results accurately reflect the raw data of the study.

QA activities for this study:

QA Activity Date Conducted Date Findings Reported To Study Director And Management
Protocol review June 17, 2010 1 ; Oct 6, 2010 June 17, 2010; Oct 6, 2010
In-process inspection: Day 2 in-life observations Aug 26, 2010 Oct 6, 2010

Raw data audit Oct 6, 2010 Oct 6, 2010
Draft report review Oct 6, 2010 Oct 6, 2010
Final report reviewed by:

1 Dow’s “generic” protocol used for this study was revi ewed by the Quality Assurance group on this date.Page 5 of 41 EPSL Study Number Dow Study Number



### TABLE OF CONTENTS

STATEMENT OF NO DATA CO NFIDENTIALITY CLAIMS.......................................................................... 2
GOOD LABORATORY PR ACTICE COMPLIANCE STATEMENT .............................................................. 3 QUALITY ASSURANCE STATEMENT .................................................................................................... .... 4
TABLE OF CONTENTS.............................................................................................................. .................. 5 LOCAL LYMPH NODE ASSAY (LLNA) IN MICE ......................................................................................... 6
1. PURPOSE ........................................................................................................................ ...................... 6
2. SUMMARY ........................................................................................................................ ..................... 6
3. MATERIALS ...................................................................................................................... ..................... 7
4. METHODS........................................................................................................................ ...................... 9
5. PROCEDURE...................................................................................................................... ................... 9
6. EVALUATION..................................................................................................................... .................. 11
7. STATISTICAL ANALYSIS ........................................................................................................... ......... 12
8. STUDY CONDUCT.................................................................................................................. ............. 12
9. REFERE NCES ..................................................................................................................... ................ 12
10. QUALITY ASSURA NCE.............................................................................................................. ......... 13
11. AMENDMENT TO TH E PROTOCOL................................................................................................... 13
12. DEVIATIONS FROM THE FINAL PR OTOCOL ................................................................................... 13
13. FINAL REPORT AND RECORD S TO BE MAIN TAINED .................................................................... 13
14. RESULTS ........................................................................................................................ ..................... 13
15. C ONC LUSION......................................................................................................................... ............. 14
SIGNATURE ...................................................................................................................... ......................... 15
TABLE 1: PRELIMINARY TO XICITY TESTIN G SCORES........................................................................ 16
TABLE 2: INDIVIDUA L BODY WEIGHTS .............................................................................................. ... 17
TABLE 3: INDIVI DUAL DPM VALUES ................................................................................................ ...... 19
TABLE 4: STIM ULATION INDEX .................................................................................................... .......... 20
TABLE 5: INDIVIDUAL DERM AL IRRITATION SCORES......................................................................... 21
TABLE 6: PRIMARY SKIN I RRITATION SCOR ING SYSTEM.................................................................. 24
APPENDIX A: PROTOCOL .......................................................................................................... ............. 25
APPENDIX B: CERTIFIC ATE OF ANALYSIS ............................................................................................ 41Page 6 of 41 EPSL Study Number Dow Study Number



### LOCAL LYMPH NODE A SSAY (LLNA) IN MICE

PROTOCOL NO.: DOW

AGENCY: EPA (FIFRA), FDA, OECD, JMAFF and EC Directive

STUDY NUMBERS: EPSL Study Number Dow Study Number

SPONSOR: THE DOW CHEMICAL COMPANY Midland, MI 48674 USA

TEST SUBSTANCE IDENTIFICATION: Lot #:

DATE RECEIVED: August 12, 2010

EPSL REFERENCE NO.:

STUDY INITIATION DATE: August 18, 2010

DATES OF TEST: August 18 –August 31, 2010

IN-LIFE DATES: August 18 – August 31, 2010

NOTEBOOK NO.: 10-135: pages 255, 255A-260C, 261-274A, 275- 289A, 290-300D

1. PURPOSE
To determine the dermal sensiti zation potential of using the mouse local lymph node assay (LLNA).

2. SUMMARY
A dermal sen sitization test was conducted with mice to determ ine the potential for to produce sensitization after repeated topical applications.
Three concentrations of the test substance in acet one/olive oil (AOO; 4:1 v/v) or the vehicle alone were topically applied to twenty healthy female test mice (5 m ice/group) for three consecutiv e days. Three da ys after t he last appli cation, the mice were given a 2 0 μ Ci IV injection of Thymidine, [Methyl- 3 H]-. Five hours l ater, the draining (auricul ar) lymph nodes were harvested and prepared for analy sis in a scintillation counte r. The results are presented in disintegrations per minute per mouse (dpm/mouse). Each ani mal’s ears were also evaluated f or erythema and edema prior to each application and aga in on Day 6, prior to the IV injection. A positive control group (five f emale mice) was maintained under the same environmental conditions and treatedPage 7 of 41 EPSL Study Number Dow Study Number

with a 25% w/w mixture of alpha-Hexylcinnamal dehyde Technical (HCA) in AOO (4:1 v/v) in the same manner as the test animals. A table summarizing the results of the LLNA is found below:

Mean DPM Stimulation Index 2 Group 1-Vehicle Control 962.76 – Group 2- 5% Test substance 1148.78 1.19 Group 3- 10% Test substance 606.54 0.63 Group 4- 25% Test substance 732.28 0.76 Group 5- Positive Control 3525.30 3.66 Based on the results of th is study , the test substance is considered to lack der mal sensitization potential in t he LLNA at concentrations of less than or equal to 25%. Proper conduct of the LLNA was confirmed via a positive response with 25% HCA, a moderate contact sensitizer.

3. MATERIALS
A. Test Substance The test substance, identified as Lot #: was received on August 12, 2010 and was further identified w ith EPSL Reference Number The test substance was stored at room temperature. The test sa mple was soluble in a 4:1 v/v m ixture in acetone/olive oil, which made the best hom ogenous m ixture. Preli minary sam ple prepara tions conducted by EPSL indicated that mixtures in excess of 25% ( i.e., 50% and 75%) were too viscous to be applied properly, therefo re 25% was the highest concentration tested. A tis sue tearor (Tissu emiser, Model OM02006) was used on each preparation to for m a ho mogenous mixture. Documentation o f the m ethods of s ynthesis, fabrication, or derivation of the test substance is retained by the Sponsor. Determination of the stabilit y, identity, strength, purity, and composition of the test substance is the responsibility of the study Sponsor. The follow ing information related to the characterization of the test substance was provided by the Sponsor: Composition: See Certificate of Analysis in Appendix B. Physical description: Off-white solid Solubility: Not provided. Stability: Test substance was expected to be stable for the duration of testing. Expiration Date: March 06, 2012 Storage Conditions: Ambient

2 The stimulation index is derived by dividing the dpm of each experimental group by the dpm of the vehicle control

group. A stimulation index of greater than or equal to 3.0 generally indicates a positive response.Page 8 of 41 EPSL Study Number Dow Study Number

B. Thymidine, [Methyl- 3 H]- Thymidine, [Methyl- 3 H]-, Lot #201008, was received on Augus t 27, 2010 and stored refrigerated. Documentation of t he m ethods of s ynthesis, fabric ation, or deriv ation of the test substance is retained by Perkin Elmer Life and Analytical Science, Boston, MA. The following information related to the character ization of the radioisotope was provided on the Certificate of Analysis: Specific Activity: 20 Ci/mmol Molecular Weight: 242 Radioactive concentration: 37 MBq/mL; 1 mCi/mL Radiochemical Purity: > 97% (HPLC) Thymine content: < 0.5% Expiration Date: September 27, 2010
C. Positive Control Substance The positive control substance, identified as alpha-Hexylcinnamaldehyde, Te chnical, 95%, Lot #22297EJ, was received on Septem ber 24, 2009 a nd was further identified with EPSL Reference Number 090924-5H. The t est substance was stored at room temperature. Docu mentation of the methods of s ynthesis, fab rication, or d erivation of t he test subst ance are retained b y Aldr ich Chemical Co., Milwaukee, WI 53201. Composition: alpha-Hexylcinnamaldehyde – 95.89% Physical description: Yellow liquid Solubility: Soluble in mineral oil and 4:1 v/v acetone/olive oil. Stability: Substance was expected to be stable for the duration of testing. Expiration Date: September 21, 2010.
D. Animals 3.D.1 Number of Animals: 35
3.D.2 Number of Groups: 10
3.D.3 Number of Animals per Group: Preliminary Irritation (5 groups): 2 Test (3 groups): 5 Vehicle (Negative) Control: 5 Positive Control: 5
3.D.4 Sex: Female, nulliparous and non-pregnant.
3.D.5 Species/Strain: Mouse, CBA/J
3.D.6 Age/Body weight: Preliminary An imals: Young adult (12 weeks) Test and Control Animals: Young adult (9-12 weeks)/19.0-23.4 grams at experimental start.
3.D.7 Source: Receiv ed fro m Harlan, In dianap olis, IN on July 20, 2010 (Prelimin ary Irritation Groups) and August 17, 2010 (Test and Control Groups).Page 9 of 41 EPSL Study Number Dow Study Number

4. METHODS
A. Husbandry
4.A.1 Housing: The animals were individually hou sed in plastic solid bottom cages with bedding during the dosi ng and resting phases of the study . After final weighing until sacrifice, animals were housed in their resp ective dose groups in plastic cages with bedding. Bedding in the plastic, solid bottom cages w as changed at least once per week. All caging conformed to the s i ze recommendations in the m ost recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 1996).
4.A.2 Animal Room Temperature and Rela tive Humidity Ranges: 19 -22oC and 60-67%, respectively.
4.A.3 Animal Room Air Changes/Hour: 12. Airf low measurements are evaluated regularly and the records are kept on file at Eurofins PSL.
4.A.4 Photoperiod: 12-hour light/dark cycle
4.A.5 Acclimation Period: 8 or 29 days
4.A.6 Food: Purina Rodent Chow #5001 ad libitum
4.A.7 Water: Filtered tap water was supplied ad libitum by an automatic water dispensing system.
4.A.8 Contaminants: There were no known contam inants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study. Analyses of the food and water are conducted regularly and the records are kept on file at Eurofins PSL.

B. Identification
4.B.1 Cage: Each cage was identified with a cage card indicating at least the study number, identification, and sex of the animal.
4.B.2 Animal: Each animal was marked with a color code and given a se quential animal number assigned to study which constituted unique identification.

5. PROCEDURE
A. Preliminary Toxicity Testing
Four test substance concentrations and the vehicle control were used. Test substan ce concentrations of 2.5%, 5%, 10% and 25% were tested to determ ine the highest achievable level that avoids overt sy stemic toxicity and excessi ve lo cal irritation. The top dose of 25% was selected based on m aximum solubility/compatibility of the test s ubstance with the vehicle while maintaining a pipettable so lution that could be ap plied to the m ouse ear (documented in the raw data). Each group consisted of two mice. The ea rs of each mouse were scored for erythema and edema prior to dosing on Days 1, 2, 3, and prior to termination on Day 6. Twenty-five μ L of the appropriate dilution of the test s ubstance concentration or the vehicle alone was applied to the dorsum of both ears of each mouse for three consecutive days (Day s 1, 2 and 3). Application was done using an appropriate size micropipette to accurately deliver 25 μ L. The dose was gently spread as evenly as possible over th e dorsal surface of the ear using the tip of thePage 10 of 41 EPSL Study Number Dow Study Number

pipette. No treat ment was made on Days 4 and 5. On Day 6, each site was evaluated for local irritation (erythema & edema) according to th e scoring system described in Section 5.E Animals were observed daily for signs of toxicity. The Study Director and Sponsor used this data in conjunction with any pr e-existing data to select the maximum concentration to be tested. Test substance concentrations of 5%, 10% a nd 25% w/w mixtures in acetone/olive oil (AOO; 4:1 v/v) were selected for testing.
B. Selection of Animals/Dose Levels Prior to dosing, the animals were weighed and the ears wer e c hecked for any abnormalities or clinical signs of diseases or injur y. Twenty-five healthy naive female mice without pre-existing ear irritation were selected and distributed (5 mice per group) into the following test groups:
Group # Purpose Concentration 1 Vehicle 0% 2 Test Substance 5% 3 Test Substance 10% 4 Test Substance 25% 5 Positive Control Substance 25% HCA
Concentrations were selected based on toxi city, solubility, irritancy, and viscosity.

C. Sample Preparation Dilutions of the test subst ance w ere pr epar ed as w/w mixtures i n acetone/olive oil (AOO; 4:1 v/v). Concentrations of 5%, 10% and 25% were selected for the main test based on results of the preliminary screening test. A single concentration of a 25% w/w mixture of HCA in AO O (4:1 v/v) was prepared as a positive control. All dosage preparations were freshly prepared on the day of administration.

D. Test Substance Application Beginning on Day 1, a volum e of 25 μ L of the vehicle, the appropri ate test substance concentration or the positive control substance was applied to the dorsu m of both ears of each mouse once per da y for three consecutive da ys (Day s 1, 2, and 3) using a micropipette. During application, the material was gently spread as evenly as possible over the dorsal surface of the ear using the tip of the pipette.

E. Dermal Scoring Prior to each application (Days 1, 2 a nd 3), th e ea rs were eval uated for erythe ma and ede ma according to the modified Draize scoring system 3 (see Table 6 ). All ears wer e also evaluated on Day 6.

F. Thymidine, [Methyl- 3 H]- Injections On Day 6 of the study (three days after the final topi cal application) 250 μ L of sterile phosphate buffered saline (PBS, Lo t #: 0 39K8200) containi ng 20 μ Ci of Thy midine, [ Methyl- 3 H]- (Lot #201008) was injected intravenously via the tail vein of each mouse.

3 Draize, J.H., Woodward, G ., and Calvery, H.O. Methods for the study of irritation and toxicity of substances applied topically

to the skin and mucous membranes. J. Pharmacol. Exp. Ther. 1944; 82:377-390

.Page 11 of 41 EPSL Study Number Dow Study Number

G. Lymph node assessment Approximately five hours after the injection, th e draining auricular lymph nodes from all animals were excised. The lymph nodes were pooled for each individual mouse. A single cell suspension of lymph node cells (L NC) was prepar ed in PB S by gently massaging the lymph nodes between the frosted e nds of two microscope sli des over a co llection vessel. The slides were then ri nsed briefly with PBS into the vessel. The contents of the vessel were transferred to a centrifuge tube and washed with an excess of PBS and centrifuge d for approximately 10 minutes at 1750 rpm , with a g-force of 283.15 grams. This process was carried out twice. In both cases, the supernatant was decanted and discarded following each centr ifugation. After the second wash, approximately 5 mL of 5 % trichloroacetic acid (TCA, Lot #: 08 6862) was then added to the sedim ent and the tube was vortexed briefly. The DNA was then precipitated in the TCA at a pproximately 4 ° C overnight (approximately 18 hours). Following the overnight precipitation of th e DNA, the tubes were centrifuged again for approximately 10 minutes and the supernatant w as discarded. Th e resulting precipitate was resuspended using 1 m L of TCA and transferred to 10 mL of scin tillation fluid. Incorporati on of Thymidine, [ Methyl- 3 H]- was measured by Β -scintillation counting and expressed as disintegrations per minute, minus background dpm.
H. Clinical Observations All test and preliminary s creening m ice were observed for si gns of m ortality, gross toxici ty, and/or behav ioral changes daily . All test mi ce were euthanized via over dose of inhaled Isoflurane anesthetic on Day 6.

I. Body Weights All test mice were weighed on Day 1 (prior to th e first application) and prior to sacrifice on test Day 6.

6. EVALUATION The mean and standard deviation of the dpm values were calculated for each dose group. A stimulation index (SI) was derived for each expe rimental group by dividing the mean dpm o f each experimental group by the mean dpm of the vehicle control group. Any test material that produces a SI > 3 i n the LLNA is norm ally considered “positive” for der mal sensitization potential (Kimber et al. , 1994). Whil e a SI > 3 was originall y developed empirically, a robust statistical eva luation indic ated that it is an accep table practical value for haz ard identification (Basketter et al., 1 999a). Furtherm ore, by determining EC 3 va lues (esti mated concentra tion resulting in a 3-fold SI), one can com pare rel a tive sensitization potenc y of chem icals and/or formulations (Basketter et al., 1999b). While a test material that produces a SI of > 3 in the LLNA should be conside red “positive” for contact sensitization ( Kimber et al ., 1994 ), re cent opinions have suggested circumstances in wh ich t he LLNA result and sensitization potential should be fur ther considered in the cont ext of additi onal scientific judgm ent (Ryan et al., 2000; Basketter et al., 1998; Basketter et al., 2006). Based on the EC 3 values derived from the LLNA, it has been proposed that contact alle rgens can be categorized as we ak (> 10% - < 10 0%), moderate (> 1% - < 10%), strong (> 0.1% - < 1%), or extreme (< 0.1%) (ECETOC, 2003).
If applicable, the EC 3 value will be calculated to determ ine the concentration of test subst ance that induces a sti mulation index of 3.0. It is determined by linear in terpolation between tw o points of the stimulation indices axis, one above (a,b) and one below (c,d) the sti mulation indexPage 12 of 41 EPSL Study Number Dow Study Number

of 3.0. If all dose levels in duce a stimulation index greater th an 3.0 or less than 3.0, the EC 3 will not be calculated.
{EC 3 =c+[(3-d)/(b-d)] x (a-c)}
The final interpretation of the biological si gnificance of the responses wa s based on both statistical outcome and scientific judgment.

7. STATISTICAL ANALYSIS Statistical analysis was performed on t he dpm values. Significan ce was judged at p <0. 05. The treated groups and vehicle control group were co mpared using a One-Way Analysis of Varianc e (ANOVA), followed b y comparison of the treated grou ps to control b y D unnett’s t-test for multiple co mparisons. Where variances are consider ed significant ly different by Bartlett’s t est, groups were compared using a non-par ametric me thod (Kruskal-Wallis non parametric analysis of variance followed b y Dunn’s test) (INSTAT Bi ostatistics, Graph Pad Sof tware, San Diego , CA). Outlier analysis was conducte d using Grubbs test (1969).

8. STUDY CONDUCT This study was conducted at Eurofi ns Product Safety Lab’s (Eur ofins PSL) te st facility at 2394 US Highway 130, Da yton, New Jersey 08810. The Stud y Director for this stud y was Anselm o Villagran, B.S. Due to Ansel mo Villagran leav ing the company, the study w as re-assigned to Jennifer Durando, B.S. (see Section 11). The primar y scientist for this study was Alicia Adamiec, with contributions by Matthew Sorber, B.S. a nd Maryann Zakrzewski. This study was conducted to comply with the Good Laboratory Practice (GLP) regulations as defined in:

• 40 CFR 160: U.S. EPA GLP Standards: Pesticide Programs (FIFRA), 1989

• 21 CFR 58: U.S. FDA, 1987

• OECD Princi ples of GLP (as r evised in 1997) pub lished in ENV/MC/CHEM (98)17, OECD, Paris, 1998

• JMAFF GLP Standards: 11-Nousan-No. 6283, 1999

• EC Directive 2004/10/EC, Official Journal of the European Union, L50/44, 2004 and based on the following testing guidelines:

• U.S. EPA Health Effects Test Guidelines, OPPTS 870.2600

• OECD Guidelines for the Testing of Chemicals, Test No. 429

• Official Journal of the European C ommun ities, Methods for the Determination of Toxicity and Other Health Effects, Part B.42 (Ski n Sensitization: Local L ymph Nod e Assay), Commission Regulation (EC) No. 440/2008

9. REFERENCES Basketter, D. A., Gerberick, G. F. and Kimber I. (1998). Strategi es for identifying false positive responses in predictive skin sensitization tests. Food and Chemical Toxicology 36, 327-333. Basketter, D. A., Lea, L. J., Cooper, K., Stocks , J., Dickens, A., Pate, I., Dearm an R., J., and Kimber, I. (1999a). T hreshold for cla ssification as a skin sensitizer in the local ly mph node assay: a statistical evaluation. Food and Chemical Toxicology 37(12) , 1167-1174.Page 13 of 41 EPSL Study Number Dow Study Number

Basketter, D. A., Lea, L. J., Dickens, A., Bri ggs, D., Pate, I., Dearman, R. J., and Kimber, I. (1999b). A comparison of statistical approaches to t he deviation of EC 3 values from local lymph node assay dose responses. Journal of Applied Toxicology 19, 261-266. Basketter, D. A., McFadden, J., Evans, P., Ander sen, K. E., and Jowsey , I. (2006). Identific ation and classification of skin sensitizer s: identifying false positives and false n egatives. Co ntact Dermatitis 55, 268-273. ECETOC (European Centre for Ecotoxicolog y a nd To xicology of Chemicals). Contact Sensitisation: Classifi cation According to Potenc y. Technical Re port No. 87. Brussels, April 2003 (ISSN-0773-8072-87). Kimber, I., Dearman, R. J. , Scholes, E. W., and Basketter, D. A. ( 1994). The l ocal lymph node assay: developments and applications. Toxicology 93 , 13-31. Ryan, C. A., Gerberick, G. F., Cru se, L. W., Basketter, D. A., Lea, L., Blaikie, L., Dearman, R. J., Warbrick, E. V., and Kim ber, I. (2000). Activity of human contact allergens in the murine local lymph node assay. Contact Dermatitis 43, 95-102.

10. QUALITY ASSURANCE The final report was audited for agreement with th e raw data records and for compliance with the protocol, Eurofins PSL Standard Operating Pro cedures and appropriate Good Laboratory Practice Standards. Dates of inspections and audits perfo rmed during the study, and the dates of reporting of the inspection and audit findings to the Study Director and Facility Management are presented in the Quality Assurance Statement.

11. AMENDMENT TO THE PROTOCOL Due to Anselmo Villagran leaving the com pany, this study was re-assigned to Jennifer Durando. This change was required in order to reassign directorship for the study.

12. DEVIATIONS FROM THE FINAL PROTOCOL
None.

13. FINAL REPORT AND RECORDS TO BE MAINTAINED The original signed final report will be sent to the Sponsor. The ori ginal raw data, documentation, records an d the ori ginal protocol an d final report generated as a result of t his study will be sent to the Sponsor for retention fo llowing signing of the final report. Copies of the raw data, protocol an d fi nal report, along with all stud y correspondence wil l be retained b y Eurofins PSL. Eurofins PSL will m aintain these reco rds for a period of at least five years. After this time, the copies of the raw data, protoc ol and final report, along with all stud y correspondence will be destroyed.

14. RESULTS Preliminary toxicity scoring results are presented in Table 1 . Individual body weights for vehicle, test, and positive control a nimals are pr esented in Table 2 . Indivi dual dpm values are presented in Table 3 . A summary of results for vehicle, test, and positive control anim als is present ed in Table 4 . Individual dermal irritation scores are presented in Table 5 . The modified Draize Primary Skin Irritation Sc oring System is presented in Table 6 . The Protocol and the Certifi cate of Analysis are presented in Appendices A and B , respectively.Page 14 of 41 EPSL Study Number Dow Study Number

A preliminary screening s tudy was conducted with concentrations of 2.5%, 5%, 10% and 25%. The top dose of 25% was selected based on m a ximum solubility/compatibility of the test substance wit h the vehicle while maintaining a pi pettable solution that could be applied to the mouse ear (documented in the raw data). On Days 1, 2, 3 and 6, each dose site was evaluated for local irritation. Very slight erythema was noted in mice dosed at all concentrations. Based on the results of the screening ass ay, 5%, 10% and 25% were evaluated in the m ain study. All mice appeared a ctive and healthy, and ther e were no consistent tr eatment-related effects on body weight over the course of the study. All treat ment groups displayed very slight erythema on Days 3 and 6. Very slight to well-defined erythema was noted in the positive control group. Treatment of mice with 5%, 10% and 25% of resulted in stimulation index values of 1.1 9, 0. 63 an d 0.76, respectively, relative to vehicle c ontrol m ice. As a stim ulation index (SI) of greater than 3.0 was not observed, was considered negative for derm al sensitization po tential. Pro per c onduct of the LLNA was confirm ed via a positi ve response wit h 25% HCA, a moderate contact sensit izer, which e licited a proliferative re sponse with a SI value of 3.66 relative to vehicle controls.
15. CONCLUSION Based on the results of this study, is considered to lack dermal sensitization potential in the LLNA at concentrations of less than or equal to 25%. Proper conduct of the LLNA was confirm ed via a positive response with 25% HCA, a moderate contact sensitizer.Page 15 of 41 EPSL Study Number Dow Study Number



### SIGNATURE

I, the undersigned, declare that the me thods, results, and data contained in this report faithfully reflect the procedures used and raw data collected during the study.Page 16 of 41 EPSL Study Number Dow Study Number



### TABLE 1: PRELIMINARY TO XICITY TESTING SCORES

Erythema/Edema

Day

1 2 3 6 Animal No. Sex

Left Right Left Right Left Right Left Right
Group 1 –0% 4

3680 F 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

3681 F 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 Group 2 – 2.5% 5

3682 F 0/0 0/0 0/0 0/0 1/0 0/0 0/0 0/0

3683 F 0/0 0/0 0/0 0/0 0/0 0/0 0/0 1/0 Group 3- 5% 5

3684 F 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

3685 F 0/0 0/0 0/0 0/0 1/0 0/0 0/0 1/0 Group 4- 10% 5

3686 F 0/0 0/0 0/0 0/0 1/0 0/0 0/0 0/0

3687 F 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 Group 5- 25% 5

3688 F 0/0 0/0 0/0 0/0 1/0 0/0 1/0 1/0

3689 F 0/0 0/0 0/0 0/0 1/0 0/0 0/0 1/0

4 25 μL of a 4:1 v/v mixture of acetone in olive oil (AOO) was applied.

5 25 μL of the test substance was applied as w/w mixtures in 4:1 v/v mixture of AOO.Page 17 of 41 EPSL Study Number Dow Study Number



### TABLE 2: INDIVI DUAL BODY WEIGHTS

Body Weight Body Weight Gain Animal

No. Group Sex Day 1 (g) Day 6 (g) Days 1-6 (g) 3601 F 20.4 19.9 -0.5 3602 F 21.0 22.7 1.7 3603 F 20.9 22.6 1.7 3604 F 21.9 22.5 0.6 3605 Vehicle Control (4:1 Acetone in Olive Oil)

F 19.2 20.1 0.9
Mean 20.7 21.6 0.9

SD 0.99 1.43 0.91
3606 F 21.4 22.5 1.1 3607 F 22.6 23.9 1.3 3608 F 19.0 19.9 0.9 3609 F 21.5 22.2 0.7 3610 5% Test Substance

F 21.4 22.3 0.9
Mean 21.2 22.2 1.0

SD 1.32 1.44 0.23
3611 F 21.1 22.5 1.4 3612 F 22.6 23.3 0.7 3613 F 21.1 22.2 1.1 3614 F 19.0 19.9 0.9 3615 10% Test Substance

F 23.4 23.5 0.1
Mean 21.4 22.3 0.8

SD 1.69 1.44 0.49
3616 F 19.5 20.9 1.4 3617 F 21.7 22.7 1.0 3618 F 20.4 21.6 1.2 3619 F 20.8 21.7 0.9 3620 25% Test Substance

F 21.3 21.6 0.3
Mean 20.7 21.7 1.0

SD 0.85 0.64 0.42Page 18 of 41 EPSL Study Number Dow Study Number



### TABLE 2 (cont.): INDI VIDUAL BODY WEIGHTS

Body Weight Body Weight Gain Animal

No. Group Sex Day 1 (g) Day 6 (g) Days 1-6 (g) 3621 F 19.3 19.4 0.1 3622 F 21.6 22.4 0.8 3623 F 20.9 22.1 1.2 3624 F 22.1 22.7 0.6 3625 Positive Control (25% HCA in 4:1 Acetone in Olive Oil)

F 22.4 23.1 0.7
Mean 21.3 21.9 0.7

SD 1.23 1.47 0.40Page 19 of 41 EPSL Study Number Dow Study Number



### TABLE 3: INDI VIDUAL DPM VALUES

Background: 55.18

Dose Level Animal # dpm dpm minus background Average St Dev SI SI >3

3601 1013.62 958.44 3602 1653.13 1597.95 3603 658.03 602.85 3604 1042.03 986.85 0% Vehicle Control (AOO)

3605 722.90 667.72 962.76 393.91 – -

3606 1461.85 1406.67 3607 840.21 785.03 3608 1117.80 1062.62 3609 614.14 558.96 5% Test Substance

3610 1985.82 1930.64 1148.78 539.78 1.19 No

3611 694.47 639.29 3612 766.12 710.94 3613 477.87 422.69 3614 520.78 465.60 10% Test Substance

3615 849.35 794.17 606.54 158.78 0.63 No

3616 677.78 622.60 3617 753.01 697.83 3618 532.54 477.36 3619 1146.60 1091.42 25% Test Substance

3620 827.35 772.17 732.28 228.46 0.76 No

3621 2821.25 2766.07 3622 7597.61 6 7542.43 3623 2034.79 1979.61 3624 2601.93 2546.75 Positive Control (25% HCA in AOO)

3625 2846.82 2791.64 3525.30 2269.29 3.66 Yes

6 Value found to be an outlier by the Grubb’s Test; statistical outlier not excluded from analysis.Page 20 of 41 EPSL Study Number Dow Study Number



### TABLE 4: STIM ULATION INDEX

Dose Level Group # Group Mean DPM SI Sensitization Response Vehicle Control 1 962.76 – N/A 5% Test Substance 2 1148.78 1.19 Not a Sensitizer 10% Test Substance 3 606.54 0.63 Not a Sensitizer 25% Test Substance 4 732.28 0.76 Not a Sensitizer 25% HCA-Positive Control 5 3525.30** 3.66 Positive - valid study N/A= Not Applicable

** Statistically significant difference from vehicle co ntrol at p < 0.01 by Dunnett Multiple Comparisons

TestPage 21 of 41 EPSL Study Number Dow Study Number



### TABLE 5: INDIVIDUAL DE RMAL IRRITA TION SCORES

Group 1 – Vehicle Control 7
Erythema/Edema

Days

1 2 3 6 Animal

No. Sex

Left Right Left Right Left Right Left Right

3601 F 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

3602 F 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

3603 F 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

3604 F 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

3605 F 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

Group 2 – 5% 8
Erythema/Edema

Days

1 2 3 6 Animal

No. Sex

Left Right Left Right Left Right Left Right

3606 F 0/0 0/0 0/0 0/0 1/0 0/0 1/0 0/0

3607 F 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

3608 F 0/0 0/0 0/0 0/0 1/0 1/0 1/0 1/0

3609 F 0/0 0/0 0/0 0/0 1/0 0/0 0/0 0/0

3610 F 0/0 0/0 0/0 0/0 1/0 1/0 1/0 1/0

7 25 μL of a 4:1 v/v mixture of acetone in olive oil (AOO) was applied.

8 25 μL of the test substance was applied as a w/w mixture in a 4:1 v/v mixture of AOO.Page 22 of 41 EPSL Study Number Dow Study Number



### TABLE 5 (cont.): INDIVIDUAL DERMAL IRRITATION SCORES
Group 3 – 10% 9
Erythema/Edema

Days

1 2 3 6 Animal

No. Sex

Left Right Left Right Left Right Left Right

3611 F 0/0 0/0 0/0 0/0 1/0 1/0 1/0 0/0

3612 F 0/0 0/0 0/0 0/0 0/0 1/0 0/0 0/0

3613 F 0/0 0/0 0/0 0/0 1/0 1/0 1/0 0/0

3614 F 0/0 0/0 0/0 0/0 1/0 1/0 1/0 1/0

3615 F 0/0 0/0 0/0 0/0 1/0 1/0 0/0 1/0

Group 4 – 25% 9
Erythema/Edema

Days

1 2 3 6 Animal

No. Sex

Left Right Left Right Left Right Left Right

3616 F 0/0 0/0 0/0 0/0 1/0 1/0 1/0 1/0

3617 F 0/0 0/0 0/0 0/0 1/0 1/0 1/0 1/0

3618 F 0/0 0/0 0/0 0/0 1/0 1/0 1/0 1/0

3619 F 0/0 0/0 0/0 0/0 0/0 1/0 0/0 0/0

3620 F 0/0 0/0 0/0 0/0 1/0 0/0 1/0 0/0

9 25 μL of the test substance was applied as a w/w mixture in a 4:1 v/v mixture of AOO.Page 23 of 41 EPSL Study Number Dow Study Number



### TABLE 5 (cont.): INDIVIDUAL DERMAL IRRITATION SCORES
Group 5 – Positive Control 10

Erythema/Edema

Days

1 2 3 6 Animal

No. Sex

Left Right Left Right Left Right Left Right

3621 F 0/0 0/0 1/0 1/0 2/0 1/0 2/0 11 1/0 11

3622 F 0/0 0/0 1/0 1/0 2/0 2/0 1/0 1/0

3623 F 0/0 0/0 1/0 1/0 2/0 2/0 2/0 11 1/0

3624 F 0/0 0/0 1/0 1/0 2/0 2/0 2/0 2/0

3625 F 0/0 0/0 1/0 1/0 1/0 2/0 2/0 11 2/0 11

10 25 μL of a 25% w/w mixture of HCA in a 4:1 v/v mixture of AOO.

11 White crust like residue at the dose site.Page 24 of 41 EPSL Study Number Dow Study Number



### TABLE 6: PRIMARY SKIN IRRITATION SCORING SYSTEM 12

Evaluation of Skin Reactions Value

Erythema and eschar formation:
No erythema.................................................................................................................... ................... 0
Very slight erythema (barely perceptible) ...................................................................................... ... 1
Well-defined erythema.......................................................................................................... ............. 2
Moderate to severe erythema .................................................................................................... ......... 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) .................................. 4

Edema formation:

No edema ....................................................................................................................... .................... 0
Slight edema (barely perceptible) .............................................................................................. ........ 1
Marked edema (swelling is obvious) ............................................................................................. .... 2

12 Draize, et al (modified).Page 25 of 41 EPSL Study Number Dow Study Number



### APPENDIX A: PROTOCOLPage 26 of 41 EPSL Study Number Dow Study NumberPage 27 of 41 EPSL Study Number Dow Study NumberPage 28 of 41 EPSL Study Number Dow Study NumberPage 29 of 41 EPSL Study Number Dow Study NumberPage 30 of 41 EPSL Study Number Dow Study NumberPage 31 of 41 EPSL Study Number Dow Study NumberPage 32 of 41 EPSL Study Number Dow Study NumberPage 33 of 41 EPSL Study Number Dow Study NumberPage 34 of 41 EPSL Study Number Dow Study NumberPage 35 of 41 EPSL Study Number Dow Study NumberPage 36 of 41 EPSL Study Number Dow Study NumberPage 37 of 41 EPSL Study Number Dow Study NumberPage 38 of 41 EPSL Study Number Dow Study NumberPage 39 of 41 EPSL Study Number Dow Study NumberPage 40 of 41 EPSL Study Number Dow Study NumberPage 41 of 41 EPSL Study Number Dow Study Number



### APPENDIX B: CERTIFICATE OF ANALYSIS