Registration Dossier

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March 2014 to 18 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

No further details specified in the study report.

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/J Rj mice
Source: ELEVAGE JANVIER
Route des Chènes Secs B.P. 4105
53940 LE GENEST-ST-ISLE, France
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group (Experiments I-II.) or 5 animals / group (Experiment III.)
Sex: Female, nulliparous, non pregnant
Age of animals at starting: 10 weeks old (Experiment I.); 11 weeks old (Experiment II.); 9 weeks old (Experiment III.). (Animals were age-matched, within one week in each test)
Body weight range at starting: 20.8-21.7 grams (Experiment I.); 20.1-22.5 grams (Experiment II.); 21.6-24.5 grams (Experiments III.) (The weight variation in animals of the study did not exceed ± 20 % of the mean weight in each test.)
Acclimatization time: at least 5 days
Note: In the preliminary experiment, mice of 8 weeks of age (18.7-19.9 grams) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 17.8 - 25.3°C
Relative humidity: 30 - 75 %
Ventilation: 15-20 air exchange/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.
Room/Cabinet (non-radioactive phase): 244/3 (preliminary experiment); 244/4 (Experiment I.); 242/1 (Experiment II.); 244/3 (Experiment III.)
Room/Cabinet (radioactive phase): 139 – 140

Food and feeding
Animals received ssniff SM R/M-15mm "Autoclavable complete diet for rats and mice – breeding and maintenance" (Batch number: 186 0298, Expiry date: 31 May 2014; and Batch number: 672 1467, Expiry date: 30 November 2014) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Water supply
Animals received tap water from the municipal supply from 500 mL bottle, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at CiToxLAB Hungary Ltd.

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-FASERN Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, 73494 Rosenberg, Germany) was available to animals during the study. Nest building material (Cotton Rolls produced by AsBe-wood GmbH, Berta-von-Suttner-Allee 4, 21614 Buxtehude, Germany or by DC Dental Central Grosshandelges. mbH, Carl-Zeiss Str. 2, D-22946 Trittau, Germany) was also provided to the animals.

Identification and randomisation
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of CiToxLAB Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number.
The animals were randomised and allocated to the experimental groups in each experiment. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

Vehicle:

methyl ethyl ketone

Concentration:

50%, 25% and 10%

No. of animals per dose:

2 animals/dose

Details on study design:

Dose Selection and Justification of Dose Selection
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/J Rj mice using two doses (2 animals/dose) at test item concentrations of 50 and 25% (w/v) in MEK. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.
In the Preliminary Irritation / Toxicity Test, no mortality or clinical signs of systemic toxicity were observed. Test item precipitate was observed on the ears of the animals in the 50 and 25% (w/v) dose groups on Days 1-3. No marked body weight loss (>5%) was observed in the experimental animals.
Ear thickness of the animals was measured by using a thickness gauge on Days 1, 3 and 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. Both ears of each mouse were observed for erythema and scored.
The draining auricular lymph nodes of the animals were visually examined: the appearance of the lymph nodes was normal in the test item treated groups (subjective judgement by analogy with observations of former experiments).
Based on these results, 50% (w/v) dose was considered to be acceptable as the highest examined concentration for the main test.

Topical application
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

OBSERVATIONS
Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the each test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the lymph nodes were collected in Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. In Experiments I-II., the nodes of mice from each test group were pooled; in Experiment III., the lymph nodes of each animal were processed individually.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C.
After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes (Experiments I-II.) or for each animal (Experiment III.).

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules.
After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed. Pellets were resuspended in 1 mL of 5% (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5% (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

DPM was measured for each sample (for each pooled group of nodes in Experiments I-II., or for each animal in Experiment III.). The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5% (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) for each sample following the industry standard for data presentation.
Stimulation index (SI = mean DPN value of a treated group divided by the mean DPN value of the negative control group) for each treatment group was also calculated in all experiments. A stimulation index of 3 or greater is an indication of a positive result.
In Experiment III., the use of the individual approach to calculate SI made the use of a statistical analysis available*. The statistical analysis was performed using the SPSS/PC+ (4.0.1) software package. The heterogeneity of variance between groups was checked by Bartlett's test for the measured DPM values. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the result was positive, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using Mann-Whitney U-test.

Positive control results:

No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index values of 26.5, 4.4 and 15.2 in Experiments I, II and III, respectively) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

Cellular proliferation data / Observations:

Based on the results of the main test (Experiment I.), an additional experiment (Experiment II.) was performed for clarification. The additional experiment was performed using the same treatment groups and same experimental conditions.
Based on the results of the second main test, another additional experiment (Experiment III.) was performed for clarification. The additional experiment was performed using the same treatment groups (but 5 animals/group) and same experimental conditions except that individual approach was used in the radioactive proliferation measurement.

In each experiment, the appearance of the lymph nodes was normal in the negative (vehicle) control group and for most of the animals in the test item treated groups. Larger than normal lymph nodes was observed in the positive control groups, and slightly enlarged lymph nodes were recorded for one test item treated animal in the 50% (w/v) dose group in Experiment I. (subjective judgement by analogy with observations of former experiments).
In Experiment I., the stimulation index values were 3.1, 1.0 and 3.3 at concentrations of 50, 25 and 10% (w/v), respectively.
In Experiment II., the stimulation index values were 1.1, 0.7 and 1.1. at concentrations of 50, 25 and 10% (w/v), respectively.
In Experiment III., the stimulation index values were 0.7, 0.9 and 1.1 at concentrations of 50, 25 and 10% (w/v), respectively.

CLINICAL OBSERVATIONS
In Experiment I., no mortality was observed and no signs of systemic toxicity were detected. Test item precipitate / minimal amount of test item precipitate was detected on the ears of the animals in the 50, 25 and 10% (w/v) dose groups on Days 1-3.
In Experiment II., no mortality was observed and no signs of systemic toxicity were detected. Test item precipitate / minimal amount of test item precipitate was detected on the ears of one or more animals in the 50, 25 and 10% (w/v) dose groups on Days 1-3.
In Experiment III., no mortality was observed and no signs of systemic toxicity were detected. Test item precipitate / minimal amount of test item precipitate was detected on the ears of one or more animals in the 50, 25 and 10% (w/v) dose groups on Days 1-3.

BODY WEIGHT MEASUREMENTS
In Experiment I., no marked body weight loss (>5%) was recorded for the experimental animals except of one negative control animal and one animal in the 50% (w/v) dose group, but these values were considered as individual variability and they did not affect the results of the study.
In Experiment II., no marked body weight loss (>5%) was recorded for the experimental animals except of one animal in the 50 and 10% (w/v) dose groups and one animal in the positive control group. However, these values were considered as individual variability and they did not affect the results of the study.
In Experiment III., no marked body weight loss (>5%) was recorded for the experimental animals except of one animal in the 50 and 10% (w/v) dose groups. However, these values were considered as individual variability and they did not affect the results of the study.

Individual Body Weights for all Animals with group Means (Experiment I)

Animal Number

Identity Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight* (g)

Change # (%)

5641

5651

5687

5691

1

2

3

4

Negative (vehicle) control (MEK)

Mean

21.2

21.6

21.3

20.9

21.3

19.9

22.1

21.0

21.1

21.0

-6.1

2.3

-1.4

1.0

-1.1

5640

5649

5654

5706

5

6

7

8

SH-0850

50% (w/v)

in MEK

Mean

21.1

21.7

21.1

20.9

21.2

21.3

22.5

21.9

19.2

21.2

0.9

3.7

3.8

-8.1

0.1

5715

5656

5659

5646

9

10

11

12

SH-0850

25% (w/v)

in MEK

Mean

21.6

21.1

21.6

20.8

21.3

20.9

21.8

21.5

20.3

21.1

-3.2

3.3

-0.5

-2.4

-0.7

5662

5643

5653

5688

13

14

15

16

SH-0850

10% (w/v)

in MEK

Mean

21.2

21.5

21.6

20.8

21.3

20.4

21.9

20.7

20.9

21.0

-3.8

1.9

-4.2

0.5

-1.4

5676

5672

5669

5663

17

18

19

20

Positive control

25% (w/v) HCA

in MEK

Mean

21.6

21.2

21.4

20.9

21.3

23.9

23.2

22.8

21.4

22.8

10.6

9.4

6.5

2.4

7.3

*: Terminal body weights were measured on Day 6

#: (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

Individual Body Weights for all Animals with group Means (Experiment II)

Animal Number

Identity Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight* (g)

Change # (%)

6278

6333

6345

6328

1

2

3

4

Negative (vehicle) control (MEK)

Mean

22.4

21.2

20.4

20.6

21.2

21.4

22.1

22.0

20.8

21.6

-4.5

4.2

7.8

1.0

2.1

6318

6343

6321

6334

5

6

7

8

SH-0850

50% (w/v)

in MEK

Mean

22.5

20.9

20.1

20.3

21.0

22.4

21.4

18.9

20.7

20.9

-0.4

2.4

-6.0

2.0

-0.5

6320

6329

6326

6336

9

10

11

12

SH-0850

25% (w/v)

in MEK

Mean

22.5

20.8

20.1

20.7

21.0

23.0

21.4

21.8

21.4

21.9

2.2

2.9

8.5

3.4

4.2

6341

6323

6331

6339

13

14

15

16

SH-0850

10% (w/v)

in MEK

Mean

21.5

21.4

20.9

21.6

21.4

20.6

22.2

20.8

19.4

20.8

-4.2

3.7

-0.5

-10.2

-2.8

6324

6340

6346

6325

17

18

19

20

Positive control

25% (w/v) HCA

in MEK

Mean

21.5

21.6

21.4

21.0

21.4

19.4

22.8

22.4

21.5

21.5

-9.8

5.6

4.7

2.4

0.7

*: Terminal body weights were measured on Day 6

#: (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

Individual Body Weights for all Animals with group Means (Experiment I)

Animal Number

Identity Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight* (g)

Change # (%)

6447

6459

6487

6496

6451

1

2

3

4

5

Negative (vehicle) control (MEK)

Mean

24.2

23.7

22.8

22.5

21.9

23.0

23.3

22.8

23.3

22.1

22.7

22.8

-3.7

-3.8

2.2

-1.8

3.7

-0.7

6497

6456

6453

6491

6475

6

7

8

9

10

SH-0850

50% (w/v)

in MEK

Mean

23.9

23.7

23.2

22.1

22.4

23.1

22.4

23.5

22.3

22.5

22.6

-6.3

-0.8

-3.9

0.9

0.4

-1.9

6501

6454

6458

6486

6472

11

12

13

14

15

SH-0850

25% (w/v)

in MEK

Mean

23.8

21.6

23.7

23.4

23.0

23.1

24.1

21.5

23.8

22.9

22.3

22.9

1.3

-0.5

0.4

-2.1

-3.0

-0.8

6461

6476

6482

6478

6494

16

17

18

19

20

SH-0850

10% (w/v)

in MEK

Mean

24.3

23.7

22.0

22.4

23.2

22.5

23.4

23.6

22.3

22.6

22.9

-7.4

-1.3

-0.4

1.4

0.9

-1.4

6468

6485

6481

6483

6467

21

22

23

24

25

Positive control

25% (w/v) HCA

in MEK

Mean

24.5

23.1

22.7

22.2

22.5

23.0

24.9

23.6

23.5

21.9

23.5

23.5

1.6

2.2

3.5

-1.4

4.4

2.1

*: Terminal body weights were measured on Day 6

#: (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

DPM, DPN and Stimulation Index Values for all Groups (Experiment I)

Test Group Name	Measured DPM/group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5% (w/v) TCA)	34 32	-	-	-	-
Negative (vehicle) control (MEK)	1198	1165.0	8	145.6	1.0
SH-0850 50% (w/v) in MEK	3683	3650.0	8	456.3	3.1
SH-0850 25% (w/v) in MEK	1200	1167.0	8	145.9	1.0
SH-0850 10% (w/v) in MEK	3925	3892.0	8	486.5	3.3
Positive control (25% (w/v) HCA in MEK)	30858	30825.0	8	3853.1	26.5

DPM, DPN and Stimulation Index Values for all Groups (Experiment II)

Test Group Name	Measured DPM/group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5% (w/v) TCA)	38 37	-	-	-	-
Negative (vehicle) control (MEK)	2989	2951.5	8	368.9	1.0
SH-0850 50% (w/v) in MEK	3174	3136.5	8	392.1	1.1
SH-0850 25% (w/v) in MEK	2046	2008.5	8	251.1	0.7
SH-0850 10% (w/v) in MEK	3255	3217.5	8	402.2	1.1
Positive control (25% (w/v) HCA in MEK)	13018	12980.5	8	1622.6	4.4

DPM, DPN and Stimulation Index Values for all Groups (Experiment III)

Test Group Name	ID Number	Measured DPM	DPM	Number of Lymph Nodes	DPN	Group DPN	Stimulation Index
Background (5% (w/v) TCA)	-	30 35	-	-	-	-	-
Negative control (MEK)		187 335 259 141 140	154.5 302.5 226.5 108.5 107.5	2 2 2 2 2	77.3 151.3 113.3 54.3 53.8	90.0	1.0
SH-0850 50% (w/v) in MEK		102 194 198 240 93	69.5 161.5 165.5 207.5 60.5	2 2 2 2 2	34.8 80.8 82.8 103.8 30.3	66.5	0.7
SH-0850 25% (w/v) in MEK		171 248 171 202 148	138.5 215.5 138.5 169.5 115.5	2 2 2 2 2	69.3 107.8 69.3 84.8 57.8	77.8	0.9
SH-0850 10% (w/v) in MEK		272 203 159 194 286	239.5 170.5 126.5 161.5 253.5	2 2 2 2 2	119.8 85.3 63.3 80.8 126.8	95.2	1.1
Positive control (25% HCA in MEK)		1071 2776 1978 3706 4333	1038.5 2743.5 1945.5 3673.5 4300.5	2 2 2 2 2	519.3 1371.8 972.8 1836.8 2150.3	1370.2	15.2**

Note:

** = Significant (p<0.01, Mann-Whitney U-test versus negative control)

Results of the Preliminary Irritation / Toxicity Test

Individual Body Weights for all Animals with Group Means

Animal Number

Identity Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight* (g)

Change# (%)

5333

5345

1

2

50% (w/v)

Mean

19.9

19.0

19.5

20.8

19.8

20.3

4.5

4.2

4.4

5339

5350

3

4

25% (w/v)

Mean

19.2

18.7

19.0

20.2

17.8

19.0

5.2

-4.8

0.3

Notes:

*: Terminal body weights were measured on Day 6.

#: (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

Individual Ear Thickness for all Animals

Animal Number

Identity Number

Test Group Name

Ear Thickness on Day 1

(mm)

Ear Thickness on Day 3

(mm)

Ear Thickness on Day 6

(mm)

Biopsy weight on Day 6

(mg)

5333

5345

5339

5350

1

2

3

4

50% (w/v)

25% (w/v)

0.20 / 0.21

0.20 / 0.20

0.21 / 0.21

0.22 / 0.21

0.21 / 0.20

0.20 / 0.20

0.21 / 0.22

0.22 / 0.21

0.21 / 0.20

0.20 / 0.20

13.13

13.22

14.18

12.92

Notes:

Ear thickness measurements: (data of right ear) / (data of left ear)

General historical control range for biopsy: 11.92-22.53 mg. Positive response is over 28.16 mg.

Summarized Clinical Observations

Period	Group	Animal No.	Identity No.	Clinical observations
DAY 1	50% (w/v)	1	5333	Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0
	50% (w/v)	2	5345	Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0
	25% (w/v)	3	5339	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0
	25% (w/v)	4	5350	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0
DAY 2	50% (w/v)	1	5333	Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0
	50% (w/v)	2	5345	Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0
	25% (w/v)	3	5339	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0
	25% (w/v)	4	5350	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0
DAY 3	50% (w/v)	1	5333	Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0
	50% (w/v)	2	5345	Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: 0
	25% (w/v)	3	5339	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0
	25% (w/v)	4	5350	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: 0
DAY 4	50% (w/v)	1	5333	Symptom-free, ES: 0
	50% (w/v)	2	5345	Symptom-free, ES: 0
	25% (w/v)	3	5339	Symptom-free, ES: 0
	25% (w/v)	4	5350	Symptom-free, ES: 0
DAY 5	50% (w/v)	1	5333	Symptom-free, ES: 0
	50% (w/v)	2	5345	Symptom-free, ES: 0
	25% (w/v)	3	5339	Symptom-free, ES: 0
	25% (w/v)	4	5350	Symptom-free, ES: 0
DAY 6	50% (w/v)	1	5333	Symptom-free, ES: 0
	50% (w/v)	2	5345	Symptom-free, ES: 0
	25% (w/v)	3	5339	Symptom-free, ES: 0
	25% (w/v)	4	5350	Symptom-free, ES: 0

Notes:

The clinical observation of animals on the first day was performed simultaneously with the body weight measurements.

ES: Erythema score

*: test item precipitate, **: minimal amount of test item precipitate

Summarized Clinical Observations

Summarized Clinical Observations (Experiment I)

Group

Identity No.

CLINICAL OBSERVATIONS

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

Negative control

(MEK)

5641

5651

5687

5691

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

SH-0850

50% (w/v)

in MEK

5640

5649

5654

5706

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

Symptom-free

SH-0850

25% (w/v)

in MEK

5715

5656

5659

5646

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

Symptom-free

SH-0850

10% (w/v)

in MEK

5662

5643

5653

5688

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

Symptom-free

Positive control (25% (w/v) HCA

in MEK)

5676

5672

5669

5663

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

Notes:

BT: before treatment, AT: after treatment

*: test item precipitate, **: minimal amount of test item precipitate

Summarized Clinical Observations (Experiment II)

Group

Identity No.

CLINICAL OBSERVATIONS

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

Negative control

(MEK)

6278

6333

6345

6238

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

SH-0850

50% (w/v)

in MEK

6318

6343

6321

6334

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

Symptom-free

SH-0850

25% (w/v)

in MEK

6320

6329

6326

6336

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

Symptom-free

SH-0850

10% (w/v)

in MEK

6341

6323

6331

6339

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

Positive control (25% (w/v) HCA

in MEK)

6324

6340

6346

6325

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

Notes:

BT: before treatment, AT: after treatment

*: test item precipitate, **: minimal amount of test item precipitate

Summarized Clinical Observations (Experiment I)

Group

Identity No.

CLINICAL OBSERVATIONS

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

Negative control

(MEK)

6447

6459

6487

6496

6451

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

SH-0850

50% (w/v)

in MEK

6497

6456

6453

6491

6475

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

Symptom-free

SH-0850

25% (w/v)

in MEK

6501

6454

6458

6486

6472

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

Symptom-free

SH-0850

10% (w/v)

in MEK

6461

6476

6482

6478

6494

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free

Symptom-free

Positive control (25% (w/v) HCA

in MEK)

6468

6485

6481

6483

6467

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

Notes:

BT: before treatment, AT: after treatment

*: test item precipitate, **: minimal amount of test item precipitate

Historical Control Data

(Updated in 04 July 2013)

Historical Control Data of the Positive and Negative Controls

	Vehicles
	Acetone-Olive oil (AOO)			1% Pluronic PE9200 in water			Methyl ethyl ketone (MEK)
	DPN values		SI value	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	172.6	1519.0	10.7	118.63	839.8	8.1	118.8	727.6	8.5
Range: Min: Max:	36.4 586.9	304.9 3300.5	3.2 29.2	21.4 469.6	190.6 3069.4	3.1 55.1	72.2 232.5	412.6 1042.6	4.9 12.0
Number of cases	100	72	72	82	55	55	4	2	2

	Vehicles
	N,N-Dimethylformamide (DMF)			Dimethyl sulfoxide (DMSO)			n-Hexane: Olive Oil ((HOO)
	DPN values		SI value	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	151.2	1917.6	13.5	249.4	2166.8	9.5	125.9	914.3	6.7
Range: Min: Max:	20.8 423.1	350.9 4438.9	3.8 75.7	101.6 553.3	1052.8 5291.3	4.2 24.1	81.1 165.9	490.0 1296.4	5.0 8.3
Number of cases	91	43	43	27	19	19	10	5	5

	Vehicles
	Propylene glycol (PG)			Absolute ethanol: Distilled water 70:30 mixture (EtOH)
	DPN values		SI value	DPN Values			SI values
	Control	HCA 25%	HCA 25%	Control	HCA 10%	HCA 25%	HCA 10%	HCA 25%
Average	131.8	1317.3	12.2	123.1	1264.2	3805.0	17.3	36.8
Range: Min: Max:	50.6 288.8	510.4 2280.2	3.7 27.9	52.6 357.6	1214.8 1313.5	2178.8 9207.1	17.1 17.4	25.7 54.3
Number of cases	22	16	16	6	2	5	2	5

HCA = α-Hexylcinnamaldehyde

SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group.

DPN (Disintegrations Per Node) = DPM (Disintegrations Per Minute) divided by the number of lymph nodes.

In case of individual approach, SI values were calculated from the mean DPN values of the group.

Interpretation of results:

GHS criteria not met

Conclusions:

The study required some repeat experiments; in Experiment I. the dose results did not follow a conventional dose response, in Experiment II. the negative control value was outside the expected historical range. In Experiment III., the stimulation index values were 0.7, 0.9 and 1.1 at concentrations of 50, 25 and 10% (w/v), respectively.
In conclusion, under the conditions of the present assay SH-0850, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Executive summary:

The aim of this study was to determine the skin sensitisation potential of SH-0850 following dermal exposure. The study was performed with vertebrate animals as no regulatory in vitro alternative is available. The minimum number of animals was used, corresponding to the regulatory guidelines being followed.

Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline, the test item was tested for formulation compatibility in Methyl ethyl ketone (MEK). The highest achievable concentration was 50% (w/v).

The Preliminary Irritation / Toxicity Test was performed in CBA/J Rj mice using two doses (50 and 25% (w/v) concentrations) in the selected vehicle. Based on the observations recorded in the preliminary test, the 50% (w/v) concentration was selected as top dose for the main tests.

In the main tests, female CBA/J Rj mice were allocated to five groups (in Experiments I-II. four animals were used in each group; in Experiment III. five animals were used for each group):

- three groups received SH-0850 (formulated in MEK) at 50, 25 and 10% (w/v) concentrations,

- the negative control group received the vehicle (MEK),

- the positive control group received 25% (w/v) HCA (dissolved in MEK).

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality or signs of systemic toxicity were observed in the performed experiments. Test item precipitate / minimal amount of test item precipitate was detected on the ears of one or more animals in the 50, 25 and 10% (w/v) dose groups on Days 1-3 in each experiment. There was no treatment related effect on the body weight in any of the experiments. There was no indication of irritation at the site of application.

The study required some repeat experiments; in Experiment I. the dose results did not follow a conventional dose response, in Experiment II. the negative control value was outside the expected historical range. In Experiment III., the stimulation index values were 0.7, 0.9 and 1.1 at concentrations of 50, 25 and 10% (w/v), respectively.

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response (stimulation index value of 26.5, 4.4 and 15.2 in Experiments I, II, and III, respectively) was noted for the positive control chemical, this result confirmed the validity of the assay.

In conclusion, under the conditions of the present assay, SH-0850, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.