Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 July 2016 to 05 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 429 “Skin Sensitisation: Local Lymph Node Assay”(22 July 2010)
Deviations:
yes
Remarks:
see "Any other information" for details
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008, B.42. “Skin Sensitisation: Local Lymph Node Assay” (Official Journal L142, 31/05/2008) amended by Commission Regulation (EU) No 640/2012 of 6 July 2012
Deviations:
yes
Remarks:
see "Any other information" for details
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

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: 8 weeks old (age-matched, within one week)
Body weight range at starting: 18.3 – 21.2 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 7 days
Note: In the Preliminary Experiment, mice of 12 weeks of age (22.9-25.0 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: 18.6 – 25.6°C
Relative humidity: 30 - 80 %
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” produced by ssniff Spezialdiäten GmbH, 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.
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 was available to animals during the study. Certified 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.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The formulation at 50% (w/v) using AOO as vehicle was suitable for the test.
No. of animals per dose:
Preliminary Irritation/Toxicity Test: 2 animals/dose
Main Test: 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 vehicle was assessed: 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 formulation at 50% (w/v) using AOO as vehicle was suitable for the test.
The 50% (w/v), 25% (w/v), 10% (w/v) and 5% (w/v) formulations appeared to be solutions 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 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).
The Preliminary Irritation/Toxicity Test 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. 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, 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 (see “Any other information” for Erythema Scoring table). 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 Test, no mortality was observed. No test item precipitate was observed on the ears of the animals.
Marked body weight loss (>5% decrease) was detected for both animals in the 100% (undiluted) and 50 % (w/v) groups. The mean body weight loss of these groups was more than 5 %. There were no indications of any irritancy at the site of application.
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. The ear thickness values and ear punch weights were within the acceptable range.
The draining auricular lymph nodes of the animals were visually examined: they were slightly enlarged than normal for both animals of the 100% (undiluted) and for one animal of the 50% (w/v) dose groups and they were normal for the other animal of the 50% (w/v) dose group (subjective judgement by analogy with observations of former experiments).
Taking into account these results, the body weight difference was close to the 5% weight loss in 2 animals of the 100% (undiluted) and in 2 animals of the 50% (w/v) dose group. With the small group sizes in the preliminary test, it is difficult to be certain where the threshold for systemic toxicity exists; hence, to try to be sure of a valid main study and according to the Study Plan, five dose groups will be used in the main experiment so there should be three acceptable dose groups for evaluation. Therefore, 100% (undiluted), 50% (w/v), 25% (w/v), 10% (w/v) and 5% (w/v) doses will be examined in 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.

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 of each mouse 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 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 the 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 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.

EVALUATION OF THE RESULTS
The proliferative response of lymph node cells from the lymph nodes of each individual animal is expressed as radioactive disintegrations per minute (DPM) per animal. 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 disintegrations per node (DPN = DPM divided by the number of lymph nodes) for each animal following the industry standard for data presentation.
The stimulation index (SI = mean DPN of treated group divided by mean DPN of the appropriate control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
The use of the individual approach to calculate the SI made the use of a statistical analysis available.

Interpretation of Results
The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
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.
Note: In the report, the mean DPM values are shown, but all the data of the individual measurements are kept and archived in the raw data binder.
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 intergroup differences. Where significant heterogeneity was found, the normal distribution of data was examined by the Kolmogorow-Smirnow test. In the case of no 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 the Mann-Whitney U-test.

Results and discussion

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 12.0) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range. Each treated and control group included 4 animals.

In vivo (LLNA)

Results
Key result
Parameter:
EC3
Value:
17.5
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 on the ears of the animals. There were no indications of any irritancy at the site of application on the experimental animals.

BODY WEIGHT MEASUREMENT
No treatment related effects were observed on the mean body weight changes in the main study.

PROLIFERATION ASSAY
The appearance of the lymph nodes was larger than normal for all animals of the 100% (undiluted) dose group and for three animals of the 50% (w/v) dose group and in the positive control group. They were slightly enlarged than normal for one animal of the 50% (w/v) dose group and for all animals of the 25% (w/v) dose group, and they were normal for all animals of the 10% (w/v) and 5% (w/v) dose groups and in the negative control group.
The stimulation index values were 7.0; 6.7; 4.4; 1.6 and 2.2 at concentrations of 100% (undiluted), 50% (w/v) 25 % (w/v), 10% (w/v) and 5% (w/v) respectively.

Any other information on results incl. tables

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#(%)

2292

2314

2306

2312

1

2

3

4

Negative (vehicle) control

AOO

 

 

Mean

21.2

19.6

19.2

18.9

19.7

22.5

20.4

20.2

20.6

20.9

6.1

4.1

5.2

9.0

6.1

2377

2295

2305

2311

5

6

7

8

1,10-decanediyl bis methacrylate

100% (undiluted)

 

 

Mean

21.2

19.6

19.2

18.6

19.7

21.0

20.1

20.3

19.9

20.3

-0.9

2.6

5.7

7.0

3.6

2293

2378

2304

2379

9

10

11

12

1,10-decanediyl bis methacrylate

50 (w/v)% in AOO

 

 

Mean

21.2

19.2

19.8

18.8

19.8

22.0

21.3

21.4

19.8

21.1

3.8

10.9

8.1

5.3

7.0

2299

2307

2316

2300

13

14

15

16

1,10-decanediyl bis methacrylate

25 (w/v)% in AOO

 

 

Mean

21.1

20.6

19.8

18.3

20.0

22.0

20.9

21.2

19.2

20.8

4.3

1.5

7.1

4.9

4.4

2308

2302

2369

2376

17

18

19

20

1,10-decanediyl bis methacrylate

10 (w/v)% in AOO

 

 

Mean

20.8

20.0

19.4

19.6

20.0

21.1

20.8

21.0

21.2

21.0

1.4

4.0

8.2

8.2

5.5

2320

2353

2317

2351

21

22

23

24

1,10-decanediyl bis methacrylate

5 (w/v)% in AOO

 

 

Mean

20.4

20.3

20.2

18.9

20.0

21.6

20.2

21.8

19.1

20.7

5.9

-0.5

7.9

1.1

3.6

2355

2318

2366

2354

25

26

27

28

Positive control

25 (w/v)% HCA in AOO

 

 

Mean

20.5

20.4

19.0

19.4

19.8

21.9

20.7

20.5

19.1

20.6

6.8

1.5

7.9

-1.5

3.7

Notes:

*: Terminal body weights were measured on Day 6

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

 

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name

Identity Number

Mean DPMNote

Number of lymph nodes

DPN

Mean DPN

Stimulation Index

Background

(5% (w/v) TCA)

-

-

-

-

-

-

Negative control

(AOO)

1

2

3

4

724.0

705.5

1552.0

476.5

2

2

2

2

362.0

352.8

776.0

238.3

432.3

1.0

1,10-decanediyl bis methacrylate

100% (undiluted)

5

6

7

8

4443.5

5096.5

5158.5

9531.5

2

2

2

2

2221.8

2548.3

2579.3

4765.8

3028.8

7.0*

1,10-decanediyl bis methacrylate

50 (w/v)% in AOO

9

10

11

12

1388.0

5409.5

9606.0

6653.0

2

2

2

2

694.0

2704.8

4803.0

3326.5

2882.1

6.7*

1,10-decanediyl bis methacrylate

25 (w/v)% in AOO

13

14

15

16

3351.5

3426.5

5367.5

3091.5

2

2

2

2

1675.8

1713.3

2683.8

1545.8

1904.6

4.4*

1,10-decanediyl bis methacrylate

10 (w/v)% in AOO

17

18

19

20

795.0

2279.0

1771.5

797.5

2

2

2

2

397.5

1139.5

885.8

398.8

705.4

1.6NS

1,10-decanediyl bis methacrylate

5 (w/v)% in AOO

21

22

23

24

2148.5

1953.5

793.0

2637.5

2

2

2

2

1074.3

976.8

396.5

1318.8

941.6

2.2*

Positive control

(25% HCA) in AOO

25

26

27

28

7507.5

9976.5

14103.0

9979.5

2

2

2

2

3753.8

4988.3

7051.5

4989.8

5195.8

12.0*

Notes:

1. NS = Not Significant (p<0.05, 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)

4. Note: The background values were in the 32 – 35 range.

 

Summarized Clinical Observations

Group

Animal No.

Identity No.

CLINICAL OBSERVATIONS

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

Negative control (AOO)

2292

 

2314

 

2306

 

2312

 

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

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

1,10-decanediyl bis methacrylate

100% (undiluted)

2377

 

2295

 

2305

 

2311

 

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: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

1,10-decanediyl bis methacrylate

50 (w/v)% in AOO

2293

 

2378

 

2304

 

2379

 

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

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

1,10-decanediyl bis methacrylate

25 (w/v)% in AOO

2299

 

2307

 

2316

 

2300

 

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

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

1,10-decanediyl bis methacrylate

10 (w/v)% in AOO

2308

 

2302

 

2369

 

2376

 

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

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

1,10-decanediyl bis methacrylate

5 (w/v)% in AOO

2320

 

2353

 

2317

 

2351

 

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

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Positive control

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

2355

 

2318

 

2366

 

2354

 

25

 

26

 

27

 

28

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

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Note:

1. BT: before treatment; AT: after treatment

 

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#(%)

2075

2084

1

2

100% (undiluted)

100% (undiluted)

Mean

24.5

24.0

24.3

23.2

22.8

23.0

-5.3

-5.0

-5.2

2067

2074

3

4

50% (w/v)

50% (w/v)

Mean

25.0

22.9

24.0

23.7

21.2

22.5

-5.2

-7.4

-6.3

Notes:

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

2.#= (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)

R

L

R

L

R

L

2075

2084

2067

2074

1

2

3

4

100% (undiluted)

100% (undiluted)

50% (w/v)

50% (w/v)

0.21

0.21

0.21

0.21

0.21

0.22

0.21

0.21

0.23

0.23

0.22

0.22

0.23

0.23

0.21

0.22

0.21

0.21

0.22

0.22

0.22

0.22

0.21

0.22

17.34

16.78

15.67

16.20

Note:

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

2. R: Right; L: Left

 

Summarized Clinical Observations

Period

Group

Identity No.

Animal No.

Clinical observations

DAY 1

100% (undiluted)

1

2075

Before treatment: symptom-free, ES: 0

After treatment: symptom free, ES: 0

2

2084

Before treatment: symptom-free, ES: 0

After treatment: symptom free, ES: 0

50% (w/v)

3

2067

Before treatment: symptom-free, ES: 0

After treatment: symptom free, ES: 0

4

2074

Before treatment: symptom-free, ES: 0

After treatment: symptom free, ES: 0

DAY 2

100% (undiluted)

1

2075

Before treatment: symptom-free, ES: 0

After treatment: symptom free, ES: 0

2

2084

Before treatment: symptom-free, ES: 0

After treatment: symptom free, ES: 0

50% (w/v)

3

2067

Before treatment: symptom-free, ES: 0

After treatment: symptom free, ES: 0

4

2074

Before treatment: symptom-free, ES: 0

After treatment: symptom free, ES: 0

DAY 3

100% (undiluted)

1

2075

Before treatment: symptom-free, ES: 0

After treatment: symptom free, ES: 0

2

2084

Before treatment: symptom-free, ES: 0

After treatment: symptom free, ES: 0

50% (w/v)

3

2067

Before treatment: symptom-free, ES: 0

After treatment: symptom free, ES: 0

4

2074

Before treatment: symptom-free, ES: 0

After treatment: symptom free, ES: 0

DAY 4

100% (undiluted)

1

2075

Symptom-free, ES: 0

2

2084

Symptom-free, ES: 0

50% (w/v)

3

2067

Symptom-free, ES: 0

4

2074

Symptom-free, ES: 0

DAY 5

100% (undiluted)

1

2075

Symptom-free, ES: 0

2

2084

Symptom-free, ES: 0

50% (w/v)

3

2067

Symptom-free, ES: 0

4

2074

Symptom-free, ES: 0

DAY 6

100% (undiluted)

1

2075

Symptom-free, ES: 0

2

2084

Symptom-free, ES: 0

50% (w/v)

3

2067

Symptom-free, ES: 0

4

2074

Symptom-free, ES: 0

Notes:

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

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 glycol (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

817.3

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 (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.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In conclusion, under the conditions 1,10-decanediyl bis methacrylate was shown to have sensitization potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of present 1,10-decanediyl bis methacrylate is 17.5%(w/v).
Executive summary:

The aim of this study was to determine the skin sensitisation potential of 1,10-decanediyl bis methacrylate 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 with the specific requirements of the Sponsor.

 

Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD Guideline, 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 formulation at 50% (w/v) using AOO as vehicle was suitable for the test.

 

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

 

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

- five groups received 1,10-decanediyl bis methacrylate at 100% (undiluted); or diluted in AOO at 50% (w/v), 25% (w/v), 10% (w/v) and 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 study. No test item precipitate was observed on the ears of the animals. There were no indications of any irritancy at the site of application on the experimental animals. No treatment related effects were observed on the mean body weight changes in the main study.

 

The stimulation index values were 7.0; 6.7; 4.4; 1.6 and 2.2 at concentrations of 100% (undiluted), 50% (w/v) 25 % (w/v), 10% (w/v) and 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 1,10-decanediyl bis methacrylate was shown to have sensitization potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of present 1,10-decanediyl bis methacrylate is 17.5%(w/v).

 

The following classification/labelling is triggered:

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