Registration Dossier

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 November 2016 to 22 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Principles of method if other than guideline:

Due to technical reasons, the actual relative humidity range was 23-86 % instead of 30-70% as it was indicated in the Study Plan.
The deviation is not considered not to adversely affect the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/CaOlaHsd mice
Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
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
Sex: Female, nulliparous, non pregnant
Age of animals at starting: 12 weeks old (age-matched, within one week)
Body weight range at starting: 20.0 – 22.3 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 35 days
Note: In the Preliminary Experiment I, mice of 11 weeks of age (20.1-20.8 grams) and in the Preliminary Experiment II, mice of 12 weeks of age (17.8-20.6 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: 19.0 - 24.8°C
Relative humidity: 23 - 86 %
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.

Food and feeding
Animals received ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” (Batch number: 278 5652, Expiry date: 30 November 2016 and Batch number: 141 8884, Expiry date: 31 January 2017) 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 bottles, 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).

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-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study. Nest building material was also provided for animals (ARBOCEL crinklets natural produced by J. Rettenmaier & Söhne GmbH +Co KG).

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. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100% (undiluted). The formulations at 5% (w/v), 2.5% (w/v), 1% (w/v) and 0.5 % (w/v) in AOO were suitable for treatment in this study.

No. of animals per dose:

The Preliminary Irritation/Toxicity Test I - 2 animals/dose
The Preliminary Irritation/Toxicity Test II - 2 animals/dose
Main Experiment - 4 animals/dose

Details on study design:

Formulation
The solubility of the test item was examined in a short Preliminary Compatibility Test.
The following standard OECD vehicles were assessed: AOO (acetone:olive oil 4:1 (v:v) mixture). The best vehicle taking into account the test item characteristics, its usage and the requirements of the relevant OECD guideline was considered to be AOO. The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100% (undiluted). The formulations at 5% (w/v), 2.5% (w/v), 1% (w/v) and 0.5 % (w/v) in AOO were suitable for treatment in this study. The formulations appeared to be solution by visual examination.
The test item was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)) in the Pharmacy of CiToxLAB Hungary Ltd. Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

ADMINISTRATION OF THE TEST ITEM
Dose Selection and Justification of Dose Selection
The Preliminary Irritation/Toxicity Test I was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 100% (undiluted) and 50 % (w/v) in AOO. Based on the observed ear swelling and toxicity an additional test was performed. Preliminary Irritation/Toxicity Test II using three doses (2 animals/dose) at test item concentrations of 25% (w/v), 10% (w/v) and 5% (w/v) in AOO. The preliminary experiments were 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.
The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 100 % (undiluted).
In the Preliminary Irritation / Toxicity Tests, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
During the Preliminary Irritation / Toxicity Tests, no mortality was observed. No test item precipitate was observed on the ears of the animals. In the Preliminary Experiment I systemic toxicity was observed: hunched back on Day 3 and Days 5-6, intermittent tremors on Days 3-4 were observed for both animals of the 100% (undiluted) dose group and intermittent tremors was observed for both animals of the 50% (w/v) Day 3. Peeling of skin at the treated site was observed for one animal of the 100% (undiluted) dose group and for both animals of the 50% (w/v) dose group on Days 4-6. Slightly rigid ear was observed for one animal of the 100% (undiluted) dose group and for both animals of the 50% (w/v) dose group on Days 3-6 and for the other animal of the 100% (undiluted) group on Days 3-5. Rigid ears were observed for one animal of the 100% (undiluted) dose group on Day 6. Alopecia on the top of the head was observed for both animals of the 50% (w/v) dose group on Day 6. Well-defined erythema (score 2) was observed for both animals of the 100% (undiluted) dose group on Day 3 (after treatment) and for one animal of the 50% (w/v) dose group on Days 3-4 (on Day 3 after treatment). Very slight erythema (score 1) was observed for both animals of the 100% (undiluted) and for one animal of the 50% (w/v) dose group on Days 2-6 and for the other animal of the 50% (w/v) dose group on Days 2-3 and Days 5-6.
In the Preliminary Experiment II systemic toxicity was observed: intermittent tremors were observed for both animals of the 25% (w/v) dose groups on Day 3. Slightly rigid ear was observed for both animals of the 25% (w/v), 10% (w/v) and 5% (w/v) dose groups on Day 6. Very slight erythema (score 1) was observed for both animals of the 25% (w/v) dose group on Days 2-6 and for one animal of the 10% (w/v) dose group and for both animals of the 5% (w/v) dose group on Days 2-4 and for the other animal of the 10% (w/v) dose group on Days 2-5. Alopecia around the ears was observed for one animals of the 25% (w/v) and for both animals of the 10% (w/v) dose group on Day 6.
In the Preliminary Experiment I marked body weight loss (>5% decrease) was detected for one animal of the 100 % (undiluted) dose group and for both animals of the 50% (w/v) dose group. The mean body weight loss of these dose groups were more than 5 %.
In the Preliminary Experiment II marked body weight loss (>5% decrease) was detected for one animal of the 10% (w/v) dose group and for both animals of the 5% (w/v) dose group. The mean body weight loss of these dose groups were more than 5 %.
Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after the euthanasia of the experimental animals on Day 6.
In the Preliminary Experiment I the detected ear thickness values clearly indicated excessive local irritation (= 25 %) for both animals of the 100% (undiluted) and 50% (w/v) dose groups on Day 6. The revealing ear punch weights were out of the historical control range for both animals of the 100% (undiluted) and 50% (w/v) dose groups. The increase was above the limit of positive (= 25 %) for both dose groups (except of one animal of the 100% (undiluted) dose group).
In the Preliminary Experiment II the detected ear thickness values indicated local irritation (= 25 %) for both animals of the 25% (w/v) and 10% (w/v) dose groups on Day 6. The revealing ear punch weights were out of the historical control range for both animals of the 25% (w/v) dose group and for one animal of the 10% (w/v) dose group. The increase was above the limit of positive (= 25 %) for one animal of the 25% (w/v) dose group. The revealing ear punch weights were within the acceptable range for both animals of the 5% (w/v) dose group.
The draining auricular lymph nodes of the animals were visually examined: they were larger than normal in all animals in all examined dose groups (subjective judgement by analogy with observations of former experiments).
Based on these results, 100% (undiluted), 50% (w/v), 25% (w/v) and 10% (w/v) dose groups clearly exceeded the acceptable limit; therefore these concentrations were excluded from the examined concentration series of the main test. Based on these results, 5 % (w/v) dose is selected as top dose for the main test. In the 5% (w/v) dose group the mean body weight loss was relatively close to the 5%. With the small group sizes in the preliminary tests, it is difficult to be certain where the threshold for systemic toxicity exists; hence, to try to be sure of a valid main study, four dose groups were used in the main experiment so there should be three acceptable dose groups for evaluation. Additionally, ear thickness of the experimental animals in the main test was measured by using a thickness gauge on Days 1 and 6. Additionally, on Day 6, ear thickness was determined by ear punch weight determinations, which was performed after the animals are humanely killed.

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.

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 nodes were collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. The 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 for each mouse 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 lymph nodes of each individual animal.

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 4 °C), 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 ß-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The ß-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.

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 Ear Thickness
Ear thickness of the experimental animals in the main test was determined by using a thickness gauge on Days 1 and 6 and by ear punch weight determination which was performed after the animals were humanely killed.

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

Positive control substance(s):

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

Statistics:

The use of the individual approach to calculate the 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.

Positive control results:

The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (AOO) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (stimulation index value of 15.7*) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
*Note: The stimulation index (SI) value was slightly above the upper limit of the historical positive control data, however it has not affect to the results.
Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were in within the historical control range.
Each treated and control group included 4 animals.

Cellular proliferation data / Observations:

CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the main study. No test item precipitate was observed. Slightly rigid ear was observed for three animals of the 5% (w/v) dose group on Day 3. There were no indications of any irritancy at the site of application.

EAR THICKNESS MEASUREMENT
Similarly to the preliminary experiment, ear thickness of the animals was measured using by a thickness gauge and by ear punch weight determination.
Slightly increased (=25%) ear thickness value was observed for one animal of the 5% (w/v) dose group (only left ear). Taking into account the ear thickness data of other animals of the 5% (w/v) dose group, it is considered that the outcome did not exceed the limit of excessive local irritation, therefore, this fact was considered not to adversely affect the results or integrity of the study. The revealing ear punch weights were within the historical control range in all examined dose groups.
Slightly increased ear thickness values (over the limit of positivity) were detected for three animals (one animal both ears, one animal only left ear and one animal only right ear) of the positive control group on Day 6 indicating excessive local irritation, but the biopsy weights were within the acceptable range for all animals in the positive control group. Therefore, this fact was considered not to adversely affect the results or integrity of the study.

BODY WEIGHT MEASUREMENT
No treatment related marked body weight loss (=5%) was observed on the mean body weight changes of the groups. Marked body weight loss (=5%) was observed for two animals of the 5% (w/v) dose group and the positive control group and for one animal of the 2.5% (w/v) and 1% (w/v) dose groups, however these changes were considered as animal variability.

PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative control group and in the 1% (w/v) and 0.5% (w/v) dose groups. Larger than normal lymph nodes were observed in the positive control group and in the 5% (w/v) and in the 2.5% (w/v) dose groups (with the exception of two animals in the 2.5% (w/v) dose group, these two animals had normal lymph nodes).
The stimulation index values were 12.0, 6.2, 1.8 and 1.1 at concentrations of 5%, 2.5%, 1% and 0.5 % (w/v), respectively.

INTERPRETATION OF OBSERVATIONS
The test item was a liquid, which was formulated in AOO. Since there were no confounding effects of treatment related irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulted stimulation indices observed above the threshold limit of 3 at the concentrations of 5 and 2.5 % (w/v) under these exaggerated test conditions were considered to be good evidence that 3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate is a sensitizer. Furthermore, the increase was statistically significant at these concentrations when compared to the negative control and clear dose dependence was observed.
The obtained data allow the calculation of the EC3 value according to an adequate scientific method. EC3 means the effective chemical concentration required for SI=3. The calculated EC3 value of 3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate is 1.4 % (w/v).

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^#(%)

2929

2913

2922

2894

1

2

3

4

Negative (vehicle) control AOO

Mean

20.6

20.4

21.4

22.0

21.1

21.0

21.7

20.4

21.1

21.1

1.9

6.4

-4.7

-4.1

-0.1

2906

2889

2927

2885

5

6

7

8

3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 5% (w/v) in AOO

Mean

20.3

20.6

22.0

20.8

20.9

20.1

20.9

20.5

19.6

20.3

-1.0

1.5

-6.8

-5.8

-3.0

2908

2911

2902

2916

9

10

11

12

3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 2.5% (w/v) in AOO

Mean

20.5

21.6

21.0

20.7

21.0

20.5

20.0

20.4

20.3

0.0

-7.4

-2.9

-1.4

-2.9

2900

2933

2903

2914

13

14

15

16

3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 1% (w/v) in AOO

Mean

20.8

20.2

21.8

22.3

21.3

20.3

19.5

20.3

23.1

20.8

-2.4

-3.5

-6.9

3.6

-2.3

2919

2904

2886

2895

17

18

19

20

3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 0.5% (w/v) in AOO

Mean

20.2

21.1

21.7

20.9

21.0

20.1

20.3

22.6

21.4

21.1

-0.5

-3.8

4.1

2.4

0.6

2921

2935

2947

2934

21

22

23

24

Positive control 25 (w/v) % HCA in AOO

Mean

20.0

20.7

21.2

21.0

20.7

19.8

20.4

19.8

19.7

19.9

-1.0

-1.4

-6.6

-6.2

-3.8

*: 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

Test Group Name	Animal No	Measured DPM	DPM	Number of lymph nodes	DPN	Group DPN	Stimulation Index
Background (5% (w/v) TCA)	-	25 32	-	-	-	-	-
Negative control (AOO)	2929 2913 2922 2894	248 350 348 289	214.5 316.5 314.5 255.5	2 2 2 2	107.3 158.3 157.3 127.8	137.6	1.0
3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 5 (w/v) % in AOO	2906 2889 2927 2885	5237 2894 3578 1676	5203.5 2860.5 3544.5 1642.5	2 2 2 2	2601.8 1430.3 1772.3 821.3	1656.4	12.0*
3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 2.5 (w/v) % in AOO	2908 2911 2902 2916	1054 786 2516 2652	1020.5 752.5 2482.5 2618.5	2 2 2 2	510.3 376.3 1241.3 1309.3	859.3	6.2*
3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 1 (w/v) % in AOO	2900 2933 2903 2914	567 554 610 429	535.5 520.5 576.5 395.5	2 2 2 2	266.8 260.3 288.3 197.8	253.3	1.8*
3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 0.5 (w/v) % in AOO	2919 2904 2886 2895	487 291 309 263	453.5 257.5 275.5 229.5	2 2 2 2	226.8 128.8 137.8 114.8	152.0	1.1^NS
Positive control (25% HCA) in AOO	2921 2935 2947 2934	3051 5592 3811 5002	3017.5 5558.5 3777.5 4968.5	2 2 2 2	1508.8 2779.3 1888.8 2484.3	2165.3	15.7*

Notes:

1. NS = Not Significant (Mann-Whitney U-Test versus negative control)

2. * = Significant (p<0.05, Mann-Whitney U-Test versus negative control)

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

Results of the Preliminary Irritation / Toxicity Test I

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

Animal Number

Identity Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight^*(g)

Change^#(%)

2729

2732

1

2

100% (undiluted)

Mean

20.3

20.1

20.2

19.4

18.3

18.9

-4.4

-9.0

-6.7

2734

2728

3

4

50% (w/v)

Mean

20.8

20.2

20.5

19.7

18.8

19.3

-5.3

-6.9

-6.1

Notes:

1. *: Terminal body weights were measure on Day 6

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

Individual Ear Thickness for all Animals (Preliminary Experiment I)

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)

Right

Left

Right

Left

Right

Left

2729

2732

2734

2728

1

2

3

4

100% (undiluted)

50% (w/v)

0.21

0.22

0.21

0.27

0.26

0.31

0.30

0.27

0.28

0.29

0.31

0.35

0.42

0.38

0.32

0.44

0.41

0.42

23.08

35.12

30.60

32.21

Note:

1. *: In case of ear thickness values, irritant response is considered when result is=25% above Day 1.

2. **: Historical control range: 11.92 – 22.53 mg. Postive response is over 28.16 mg (=25%).

Summarized Clinical Observations (Preliminary Experiment I)

Period	Group	Animal No.	Identity No.	Clinical observations
DAY 1	100% (undiluted)	1	2729	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
	100% (undiluted)	2	2732	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
	50% (w/v)	3	2734	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
	50% (w/v)	4	2728	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
DAY 2	100% (undiluted)	1	2729	Before treatment: symptom-free, ES: 0 After treatment: ES: 1
	100% (undiluted)	2	2732	Before treatment: symptom-free, ES: 0 After treatment: ES: 1
	50% (w/v)	3	2734	Before treatment: symptom-free, ES: 0 After treatment: ES: 1
	50% (w/v)	4	2728	Before treatment: symptom-free, ES: 0 After treatment: ES: 1
DAY 3	100% (undiluted)	1	2729	Before treatment: slightly rigid ear, ES: 1 After treatment: hunched back. intermittent tremors, slightly rigid ear, ES: 2
	100% (undiluted)	2	2732	Before treatment: slightly rigid ear, ES: 1 After treatment: hunched back. intermittent tremors, slightly rigid ear, ES: 2
	50% (w/v)	3	2734	Before treatment: slightly rigid ear, ES: 1 After treatment: intermittent tremors, slightly rigid ear, ES: 2
	50% (w/v)	4	2728	Before treatment: slightly rigid ear, ES: 1 After treatment: intermittent tremors, slightly rigid ear, ES: 2
DAY 4	100% (undiluted)	1	2729	Intermittent tremors, peeling, slightly rigid ear, ES: 1
	100% (undiluted)	2	2732	Intermittent tremors, slightly rigid ear, ES: 1
	50% (w/v)	3	2734	Peeling, slightly rigid ear, ES: 1
	50% (w/v)	4	2728	Peeling, slightly rigid ear, ES: 2
DAY 5	100% (undiluted)	1	2729	Hunched back, peeling, slightly rigid ear, ES: 1
	100% (undiluted)	2	2732	Hunched back. Slightly rigid ear, ES: 1
	50% (w/v)	3	2734	Peeling, slightly rigid ear, ES: 1
	50% (w/v)	4	2728	Peeling, slightly rigid ear, ES: 1
DAY 6	100% (undiluted)	1	2729	Hunched back, peeling, slightly rigid ear, ES: 1
	100% (undiluted)	2	2732	Hunched back, rigid ears, ES: 1
	50% (w/v)	3	2734	Peeling, slightly rigid ear, alopecia on the top of the head, ES: 1
	50% (w/v)	4	2728	Peeling, slightly rigid ear, alopecia on the top of the head, ES: 1

Notes:

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

2. ES: Erythema score

Results of the Preliminary Irritation / Toxicity Test II

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

Animal Number

Identity Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight^*(g)

Change^#(%)

2695

2697

1

2

25% (w/v)

Mean

20.4

17.8

19.1

19.4

17.5

18.5

-4.9

-1.7

-3.3

2645

2737

3

4

10% (w/v)

Mean

19.6

20.0

19.8

17.9

19.2

18.6

-8.7

-4.0

-6.3

2702

2704

5

6

5% (w/v)

Mean

18.8

20.6

19.7

16.8

19.4

18.1

-10.6

-5.8

-8.2

Notes:

1. *: Terminal body weights were measure on Day 6

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

Individual Ear Thickness for all Animals (Preliminary Experiment II)

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)

Right

Left

Right

Left

Right

Left

2695

2697

2645

2737

2702

2704

1

2

3

4

5

6

25% (w/v)

10% (w/v)

5% (w/v)

0.21

0.20

0.21

0.20

0.21

0.22

0.27

0.28

0.26

0.25

0.24

0.28

0.27

0.26

0.24

0.34

0.41

0.32

0.28

0.26

0.24

0.40

0.36

0.43

0.31

0.26

27.20

30.11

27.82

19.83

20.89

22.03

Note:

1. *: In case of ear thickness balues, irritant response is considered when result is=25% above the Day 1 value.

2. **: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (=25%)

Summarized Clinical Observations (Preliminary Experiment II)

Period	Group	Animal No.	Identity No.	Clinical observations
DAY 1	25% (w/v)	1	2695	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
	25% (w/v)	2	2697	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
	10% (w/v)	3	2645	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
	10% (w/v)	4	2737	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
	5% (w/v)	5	2702	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
	5% (w/v)	6	2704	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
DAY 2	25% (w/v)	1	2695	Before treatment: symptom-free, ES: 0 After treatment: ES: 1
	25% (w/v)	2	2697	Before treatment: symptom-free, ES: 0 After treatment: ES: 1
	10% (w/v)	3	2645	Before treatment: symptom-free, ES: 0 After treatment: ES: 1
	10% (w/v)	4	2737	Before treatment: symptom-free, ES: 0 After treatment: ES: 1
	5% (w/v)	5	2702	Before treatment: symptom-free, ES: 0 After treatment: ES: 1
	5% (w/v)	6	2704	Before treatment: symptom-free, ES: 0 After treatment: ES: 1
DAY 3	25% (w/v)	1	2695	Before treatment: ES: 1 After treatment: intermittent tremors ES: 1
	25% (w/v)	2	2697	Before treatment: ES: 1 After treatment: intermittent tremors ES: 1
	10% (w/v)	3	2645	Before treatment: ES: 1 After treatment: ES: 1
	10% (w/v)	4	2737	Before treatment: ES: 1 After treatment: ES: 1
	5% (w/v)	5	2702	Before treatment: symptom-free, ES: 0 After treatment, ES: 1
	5% (w/v)	6	2704	Before treatment: symptom-free, ES: 0 After treatment, ES: 1
DAY 4	25% (w/v)	1	2695	ES: 1
	25% (w/v)	2	2697	ES: 1
	10% (w/v)	3	2645	ES: 1
	10% (w/v)	4	2737	ES: 1
	5% (w/v)	5	2702	ES: 1
	5% (w/v)	6	2704	ES: 1
DAY 5	25% (w/v)	1	2695	ES: 1
	25% (w/v)	2	2697	ES: 1
	10% (w/v)	3	2645	ES: 1
	10% (w/v)	4	2737	Symptom-free, ES: 0
	5% (w/v)	5	2702	Symptom-free, ES: 0
	5% (w/v)	6	2704	Symptom-free, ES: 0
DAY 6	25% (w/v)	1	2695	Slightly rigid ear, ES: 1
	25% (w/v)	2	2697	Alopecia around the ears, slightly rigid ear, ES: 1
	10% (w/v)	3	2645	Alopecia around the ears, slightly rigid ear, ES: 0
	10% (w/v)	4	2737	Alopecia around the ears, slightly rigid ear, ES: 0
	5% (w/v)	5	2702	Slightly rigid ear, ES: 0
	5% (w/v)	6	2704	Slightly rigid ear, ES: 0

Notes:

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

2. ES: Erythema score

Individual Ear Thickness Values and Summarized Clinical Observations

Individual Ear Thickness for all Animals

Animal Number

Identity Number

Test Group Name

Ear Thickness on Day 1 (mm)

Ear Thickness on Day 6 (mm)^*

Biopsy weight^**on Day 6 (mg)

Right

Left

Right

Left

2929

2913

2922

2894

1

2

3

4

Negative control (AOO)

0.22

0.21

0.22

0.21

0.22

0.21

0.22

0.23

0.22

0.21

0.22

14.5

16.3

17.0

15.6

2906

2889

2927

2885

5

6

7

8

3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 5 (w/v) % in AOO

0.20

0.21

0.20

0.21

0.20

0.21

0.24

0.25

0.24

0.25

0.24

19.4

19.6

18.0

19.1

2908

2911

2902

2916

9

10

11

12

3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 2.5 (w/v) % in AOO

0.20

0.21

0.22

0.20

0.22

0.20

0.22

0.23

0.24

0.23

0.22

0.23

0.22

0.23

16.4

15.4

17.6

17.3

2900

2933

2903

2914

13

14

15

16

3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 1 (w/v) % in AOO

0.22

0.21

0.22

0.21

0.22

0.21

0.23

0.22

0.23

0.24

0.22

0.23

16.2

16.1

14.7

14.2

2919

2904

2886

2895

17

18

19

20

3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 0.5 (w/v) % in AOO

0.22

0.21

0.22

0.21

0.22

0.23

0.22

0.23

0.22

0.23

14.6

15.8

15.9

2921

2935

2947

2934

21

22

23

24

Positive control (25% HCA in AOO)

0.22

0.21

0.20

0.21

0.22

0.21

0.22

0.21

0.27

0.28

0.26

0.25

0.27

0.28

20.6

19.4

17.3

21.0

Note:

1. *: In case of ear thickness values, irritant response is considered when result is=25% above the Day 1 value.

2. **: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (=25%)

Summarized Clinical Observations

Group

Animal No.

Identity No.

CLINICAL OBSERVATION

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

Negative control (AOO)

2929

2913

2922

2894

1

2

3

4

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

3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 5 (w/v) % in AOO

2906

2889

2927

2885

5

6

7

8

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: slightly rigid ear

BT: symptom-free

AT: slightly rigid ear

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: slightly rigid ear

Symptom-free

3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 2.5 (w/v) % in AOO

2908

2911

2902

2916

9

10

11

12

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

3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 1 (w/v) % in AOO

2900

2933

2903

2914

13

14

15

16

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

3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate 0.5 (w/v) % in AOO

2919

2904

2886

2895

17

18

19

20

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 AOO)

2921

2935

2947

2934

21

22

23

24

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

Note:

1. BT: before treatment; AT: after treatment

2. ES: Erythema score

Historical Control Data

Historical Control Data of the Positive and Negative Controls for CBA/CaOlaHsd mice (2014-2015)

CBA/CaOlaHsd mice
	Vehicles
	Acetone : Olive oil 4:1 (AOO)			1% Pluronic PE9200 in water (1%Plu)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
*Average*	415.2	2922.6	7.5	197.7	1825.3	10.0
*Range: min* *max*	111.3 847.8	890.3 7674.5	3.3 15.5	23.0 680.8	154.0 6755.8	3.0 33.1
*Number of cases*	32	32	30	134	134	128
	Vehicles
	N,N-Dimethylformamide (DMF)			Dimethyl sulfoxide (DMSO)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
*Average*	244.6	2522.6	10.8	488.7	3212.1	7.8
*Range: min* *max*	140.8 505.8	1201.3 4804.6	6.3 21.3	238.5 934.6	2017.2 4877.5	3.1 14.5
*Number of cases*	21	21	21	13	13	12
	Vehicles
	Propylene gycol (PG)			Methyl ethyl ketone (MEK)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
*Average*	235.4	2371.8	10.0	260.2	4888.8	19.5
*Range: min* *max*	63.3 506.0	817.2 4978.0	6.5 14.4	183.5 383.3	2456.3 8682.5	8.9 36.3
*Number of cases*	14	14	14	9	10	10

HCA 25% = alpha-Hexylcinnamaldehyde 25% (w/v)

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

DPN (Disintegrations Per Node) – DPM (Disintegration 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:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

In conclusion, under the conditions of the present assay, 3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate, tested in a suitable vehicle, was
shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value was 1.4 %(w/v).

Executive summary:

The aim of the study was to determine the skin sensitisation potential of 3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate following dermal exposure. The study was performed with vertebrate animals as no full 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, the test item characteristics, its usage and on the recommendations of the OECD Guideline no. 429, the test item was tested for formulation compatibility in AOO (acetone:olive oil 4:1 (v:v) mixture).

The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100 % (undiluted).

The Preliminary Irritation/Toxicity Tests was performed in CBA/CaOlaHsd mice using five doses (2 animals/dose): 100 % (undiluted), 50%, 25%, 10 % and 5% (w/v) in AOO. Based on the observations recorded in the preliminary test, the 5 % (w/v) was selected as top dose for the main test.

In the main assay, twenty-four female CBA/CaOlaHsd mice were allocated to six groups of four animals each:

- four groups received 3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate (formulated in AOO) at 5% (w/v) , 2.5% (w/v), 1% (w/v) and 0.5% (w/v) concentrations,

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

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

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. 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 during the main study. No test item precipitate was observed. Slightly rigid ear was observed for three animals of the 5% (w/v) dose group on Day 3. No treatment related marked body weight loss (=5%) was observed on the mean body weight changes of the groups. Marked body weight loss (=5%) was observed for two animals of the 5% (w/v) dose group and the positive control group and for one animal of the 2.5% (w/v) and 1% (w/v) dose groups. There were no indications of any irritancy at the site of application.

The stimulation index values were 12.0, 6.2, 1.8 and 1.1 at concentrations of 5%, 2.5%, 1% and 0.5 % (w/v), respectively.

The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

In conclusion, under the conditions of the present assay, 3-{[3-(acryloyloxy)-2.2-dimethylpropanoyl]oxy}-2.2-dimethylpropyl acrylate, tested in a suitable vehicle, was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value was 1.4 %(w/v).

The following classification/labelling is triggered:

Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 6) 2015: Category 1 (sub-category 1A).