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, Methods 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

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:::: eurofins I 

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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 

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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 

Date 
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QUALITY ASSURANCE STATEMENT 

The Eurofins PSL Quality Assurance Unit has re viewed 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, 20101; 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 reviewed by the Quality Assurance group on this date. 
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TABLE OF CONTENTS

STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS.......................................................................... 2

GOOD LABORATORY PRACTICE 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. REFERENCES ..................................................................................................................................... 12

10. QUALITY ASSURANCE....................................................................................................................... 13

11. AMENDMENT TO THE PROTOCOL................................................................................................... 13

12. DEVIATIONS FROM THE FINAL PROTOCOL ................................................................................... 13

13. FINAL REPORT AND RECORDS TO BE MAINTAINED .................................................................... 13

14. RESULTS ............................................................................................................................................. 13

15. CONCLUSION...................................................................................................................................... 14

SIGNATURE ............................................................................................................................................... 15

TABLE 1: PRELIMINARY TOXICITY TESTING SCORES........................................................................ 16

TABLE 2: INDIVIDUAL BODY WEIGHTS ................................................................................................. 17

TABLE 3: INDIVIDUAL DPM VALUES ...................................................................................................... 19

TABLE 4: STIMULATION INDEX .............................................................................................................. 20

TABLE 5: INDIVIDUAL DERMAL IRRITATION SCORES......................................................................... 21

TABLE 6: PRIMARY SKIN IRRITATION SCORING SYSTEM.................................................................. 24

APPENDIX A: PROTOCOL ....................................................................................................................... 25

APPENDIX B: CERTIFICATE OF ANALYSIS ............................................................................................ 41
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LOCAL LYMPH NODE ASSAY (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 acetone/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 treated 
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with a 25% w/w mixture of alpha-Hexylcinnamaldehyde 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 

Index2

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 with 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 following 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. 
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B. Thymidine, [Methyl-3

H]- 

Thymidine, [Methyl-3

H]-, Lot #201008, was received on August 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 characterization 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 and 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 Animals: 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 dianapolis, IN on July 20, 2010 (Prelimin ary 

Irritation Groups) and August 17, 2010 (Test and Control Groups). 
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4. METHODS 

A. Husbandry 

4.A.1 Housing: The animals were individually housed in plastic solid bottom cages with 

bedding during the dosing 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 ize 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. Airflow 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 contaminants 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 ears 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 μL of the appropriate dilution of the test substance 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 μL. The 

dose was gently spread as evenly as possible over the dorsal surface of the ear using the tip of the 
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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 the 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 toxicity, solubility, irritancy, and viscosity. 

C. Sample Preparation 

Dilutions of the test subst ance w ere pr epared 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 μL 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 (Days 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 system3

 (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 μL of sterile phosphate 

buffered saline (PBS, Lo t #: 0 39K8200) containi ng 20 μCi 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. 
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G. Lymph node assessment 

Approximately five hours after the injection, the 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 PBS 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 centrifugation. 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 mice 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 ative 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 EC3 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 index 
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of 3.0. If all dose levels induce a stimulation index greater than 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 compared 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 method (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 conducted 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 primary scientist for this study was Alicia Adamiec, 

with contributions by Matthew Sorber, B.S. and 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 ommunities, 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. 
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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 EC3 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., Cruse, 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 Procedures and appropriate Good Laboratory Practice 

Standards. Dates of inspections and audits performed 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 following 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 records 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. 
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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 aximum 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 there were no consistent tr eatment-related effects on 

body weight over the course of the study. All treatment 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. 
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SIGNATURE 

I, the undersigned, declare that the methods, results, and data contained in this report faithfully reflect the 

procedures used and raw data collected during the study. 
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TABLE 1: PRELIMINARY TOXICITY TESTING SCORES 

Erythema/Edema 

Day 

Animal No. 1 2 3 6 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: INDIVIDUAL BODY WEIGHTS 

Animal Body Weight Body Weight Gain

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 0.42 
Page 18 of 41 

EPSL Study Number 

Dow Study Number 

TABLE 2 (cont.): INDIVIDUAL BODY WEIGHTS 

Animal Body Weight Body Weight Gain

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 0.40 
Page 19 of 41 

EPSL Study Number 

Dow Study Number 

TABLE 3: INDIVIDUAL 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 7597.616 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: STIMULATION 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 control at p < 0.01 by Dunnett Multiple Comparisons 

Test 
Page 21 of 41 

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Dow Study Number 

TABLE 5: INDIVIDUAL DERMAL IRRITATION SCORES 

Group 1 – Vehicle Control7

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. 
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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 Control10

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 2/011 1/011

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 2/011 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 2/011 2/011

 

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 

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Dow Study Number 

TABLE 6: PRIMARY SKIN IRRITATION SCORING SYSTEM12

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). 
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APPENDIX A: PROTOCOL 
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APPENDIX B: CERTIFICATE OF ANALYSIS