Registration Dossier

Administrative data

Description of key information

Tris(2-hydroxyethyl) isocyanurate triacrylate was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 October 2016 to 26 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 L 142, 31/05/2008) amended by Commission Regulation (EU) No 640/2012 of 6 July 2012
Deviations:
yes
Remarks:
See "Any other information" for details
Principles of method if other than guideline:
Due to technical reasons, the actual relative humidity range was 29-85 % instead of 30-70 % and the actual temperature range was 18.0- 24.7 °C instead of 22 ± 3 °C as it was indicated in the Study Plan.
The DPM measurement was performed more than once and the mean results calculated as appropriate. Measurements ended later, consequently the experiment ended later than indicated in the Study Plan.
These deviations are considered not to adversely affect the results or integrity of the study.
GLP compliance:
yes (incl. certificate)
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: 8 weeks old (age-matched, within one week)
Body weight range at starting: 19.0 – 20.7 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 9 weeks of age (19.2-19.7 grams) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health tatus was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunneltubes
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: 18.0 - 24.7°C
Relative humidity: 29 - 85 %
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. 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.
Vehicle:
dimethylformamide
Concentration:
The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100 % (w/v). The formulations at 50 and 25 % (w/v) in DMF were also suitable for treatment in this study.
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 vehicles were assessed: AOO (acetone:olive oil 4:1 (v:v) mixture) and N,N-dimethylformamide (DMF). The best vehicle taking into account the test item characteristics, its usage and the requirements of the relevant OECD guideline was considered to be DMF. The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100 % (w/v). The formulations at 50 and 25 % (w/v) in DMF were also suitable for treatment in this study.
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 was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 100 and 50 % (w/v) in DMF. 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.
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 % (w/v).
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 using Table 2 [3]. 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 or signs of systemic toxicity were observed. Alopecia around the ears or extensive alopecia was observed in the 100 % (w/v) dose group on Days 2-6 or 3-6. Test item precipitate or minimal amount of test item precipitate was observed on the ears of the animals in the 100 % (w/v) dose group on Days 2-5 or 2-6 and in the 50 % (w/v) dose group on Days 2-3. No marked body weight loss (=5%) was detected on the experimental animals.
Increased ear thickness values (more than 25%) were observed on Day 3 in the 100 % (w/v) dose group for one animal; however test item precipitate was present on the ears of the animals which may have interfered with the measurement. Slightly increased ear punch weights were detected in the 50 % (w/v) dose group, however all values in both groups were within the acceptable range.
The draining auricular lymph nodes of the animals were visually examined: they were larger than normal in both dose groups (subjective judgement by analogy with observations of former experiments).
Based on these results, 100 % (w/v) dose was selected as top dose 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.

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 samples were read more than once in the ß-scintillation counter to ensure accuracy and the mean result was calculated where applicable.
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
DPM was measured for each animal. The average of the two 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) following the industry standard for data presentation.
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 test item gave a relatively strong positive response without a clear reduction at the lowest dose level; hence the obtained data did not allow the calculation of the EC3 value of the test item (EC3 means the effective chemical concentration required for SI=3).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not specified
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 (DMF) 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 14.2) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Key result
Parameter:
SI
Value:
30.4
Test group / Remarks:
100% (w/v)
Key result
Parameter:
SI
Value:
21.2
Test group / Remarks:
50% (w/v)
Key result
Parameter:
SI
Value:
24.2
Test group / Remarks:
25% (w/v)
Cellular proliferation data / Observations:
CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the study. Alopecia around the ears was observed in the 100 % (w/v) dose group on Days 3-6 or 4-6. Test item precipitate or minimal amount of test item precipitate was observed on the ears of the animals in the 100 % (w/v) dose group on Days 1-5 or 1-4, in the 50 % (w/v) dose group on Days 2-3 and in the 25 % (w/v) dose group on Day 2.

BODY WEIGHT MEASUREMENT
No treatment related effects were observed on the body weight changes of experimental animals.

PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative control group. Larger than normal lymph nodes were observed in the positive control group and in all the test item treated dose groups (animal no.2796 had only slightly enlarged lymph nodes).
The stimulation index values were 30.4, 21.2 and 24.2 at concentrations of 100, 50 and 25 % (w/v), respectively.

INTERPRETATION OF OBSERVATIONS
The test item was a solid, which was formulated in DMF. 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 all the tested concentration under these exaggerated test conditions were considered to be good evidence that Tris(2-hydroxyethyl) isocyanurate triacrylate is a sensitizer (Figure 1). Based on these results, the test item is classified as skin sensitizer Category 1 according to the UN GHS system.
The test item gave a relatively strong positive response without a clear reduction at the lowest dose level; hence the obtained data did not allow the calculation of the EC3 value of the test item. Therefore, no sub-category according to GHS system can be determined for the test item.

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#(%)

2779

2787

2780

2761

1

2

3

4

Negative (vehicle) control DMF

 

 

 

Mean

19.4

20.4

20.0

20.4

20.1

19.6

21.7

21.0

21.2

20.9

1.0

6.4

5.0

3.9

4.1

2799

2777

2783

2790

5

6

7

8

Tris(2-hydroxyethyl) isocyanurate triacrylate

100% (w/v)

 

Mean

19.0

20.6

20.1

20.0

19.9

19.4

20.2

20.7

20.0

20.1

2.1

-1.9

3.0

0.0

0.8

2772

2798

2791

2766

9

10

11

12

Tris(2-hydroxyethyl) isocyanurate triacrylate

50% (w/v) in DMF

 

Mean

19.1

20.3

20.0

20.7

20.0

20.5

21.3

21.0

21.3

21.0

7.3

4.9

5.0

2.9

5.0

2796

2792

2771

2794

13

14

15

16

Tris(2-hydroxyethyl) isocyanurate triacrylate

25% (w/v) in DMF

 

Mean

19.3

20.1

20.5

20.2

20.0

20.7

21.8

21.5

19.9

21.0

7.3

8.5

4.9

-1.5

4.8

2762

2769

2793

2800

17

18

19

20

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

  

 

Mean

19.5

20.0

20.7

20.0

20.1

20.3

20.3

20.5

20.3

20.4

4.1

0.5

-1.0

1.5

1.3

*: 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

Identity No

Measured Total DPM (1sttime)

Measured Total DPM (2ndtime)

DPN (1sttime)

DPN (2ndtime)

Mean DPN

Group DPN

SI

Background (5% (w/v) TCA)

-

29

33

30

34

-

-

-

-

-

Negative (vehicle) control (DMF)

1

2

3

4

161

150

679

192

158

161

692

181

65.0

59.5

324.0

80.5

63.0

64.5

330.0

80.5

64.0

62.0

327.0

77.5

132.6

1.0

Tris(2-hydroxyethyl) isocyanurate triacrylate

100% (w/v) IN DMF

5

6

7

8

n.d.

5088

10015

7883

9339

4923

10180

7998

n.d.

2528.5

4992.0

3926.0

4653.5

2445.5

5074.0

3983.0

4653.5

2487.0

5033.0

3954.0

4032.0

30.4

Tris(2-hydroxyethyl) isocyanurate triacrylate

50% (w/v) in DMF

9

10

11

12

5253

4896

4847

7894

5014

4747

4692

n.d.

2611.0

2432.5

2408.0

3921.5

2491.0

2357.5

2330.0

n.d.

2551.0

2395.0

2369.0

3931.5

2811.6

21.2

Tris(2-hydroxyethyl) isocyanurate triacrylate

25% (w/v) in DMF

13

14

15

16

4118

n.d.

8577

4786

4041

8260

8775

4845

2043.5

n.d.

4273.0

2377.5

2004.5

4114.0

4371.5

2406.5

2024.0

4114.0

4322.3

2392.0

3213.1

24.2

Positive control (25% HCA in DMF)

17

18

19

20

3216

3099

4091

4893

3049

3137

4047

4902

1592.5

1534.0

2030.0

2431.0

1508.5

1552.5

2007.5

2435.0

1550.5

1543.3

2018.8

2433.0

1886.4

14.2

Notes:

1. Number of lymoh nodes was 2 in case of all animals

2. n.d.: no data due to technical reasons

3. SI: Stimulation Index

 

Due to technical reasons occasionally no DPM result is calculated by the machine on individual samples. In these cases, all samples are rerun and the

mean result for each sample is calculated (so there is a minimum of one value for every sample).

 

Results of the Preliminary Irritation / Toxicity Test

 

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

Animal Number

Identity Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight*(g)

Change#(%)

2623

2625

1

2

100% (w/v)

100% (w/v)

Mean

19.7

19.2

19.5

19.7

19.4

19.6

0.0

1.0

0.5

2618

2633

3

4

50% (w/v)

50% (w/v)

Mean

19.5

19.4

19.5

20.4

19.8

20.1

4.6

2.1

3.3

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)

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

2623

2625

2618

2633

1

2

3

4

100% (w/v)

100% (w/v)

50% (w/v)

50% (w/v)

0.22

0.21

0.22

0.21

0.21

0.21

0.22

0.22

0.34

0.26

0.24

0.22

0.32

0.25

0.23

0.23

0.23

0.23

0.24

0.21

0.24

0.23

0.23

0.23

17.38

17.80

22.42

19.42

Note:

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

 

Summarized Clinical Observations (Preliminary Experiment)

Period

Group

Animal No.

Identity No.

Clinical observations

DAY 1

100% (w/v)

2623

1

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2625

2

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

50% (w/v)

2618

3

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2633

4

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

DAY 2

100% (w/v)

2623

1

Before treatment: alopecia*, ES: 0

After treatment: alopecia**, ES: 1

2625

2

Before treatment: symptom-free*, ES: 0

After treatment: symptom-free*, ES: 0

50% (w/v)

2618

3

Before treatment: symptom-free**, ES: 0

After treatment: ES: 1

2633

4

Before treatment: symptom-free**, ES: 0

After treatment: ES: 1

DAY 3

100% (w/v)

2623

1

Before treatment: alopecia*, ES: 0

After treatment: alopecia**, ES: 1

2625

2

Before treatment: alopecia*, ES: 0

After treatment: alopecia**, ES: 1

50% (w/v)

2618

3

Before treatment: symptom-free**, ES: 0

After treatment: ES: 1

2633

4

Before treatment: symptom-free**, ES: 0

After treatment: ES: 1

DAY 4

100% (w/v)

2623

1

Extensive alopecia*, ES: 0

2625

2

Alopecia*, ES: 0

50% (w/v)

2618

3

Symptom-free, ES: 0

2633

4

Symptom-free, ES: 0

DAY 5

100% (w/v)

2623

1

Extensive alopecia*, ES: 0

2625

2

Alopecia**, ES: 0

50% (w/v)

2618

3

Symptom-free, ES: 0

2633

4

Symptom-free, ES: 0

DAY 6

100% (w/v)

2623

1

Extensive alopecia**, ES: 0

2625

2

Alopecia, ES: 0

50% (w/v)

2618

3

Symptom-free, ES: 0

2633

4

Symptom-free, ES: 0

Notes:

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

2. ES: Erythema score

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

 

 

Summarized Clinical Observations

Group

Animal No.

Identity No.

CLINICAL OBSERVATION

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

Negative control (DMF)

2779

 

2787

 

2780

 

2761

 

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

Tris(2-hydroxyethyl) isocyanurate triacrylate 100% (w/v) in DMF

2799

 

2777

 

2783

 

2790

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: alopecia*

AT: alopecia**

Alopecia**

 

Alopecia**

 

Alopecia**

 

Alopecia**

Alopecia

 

Alopecia

 

Alopecia

 

Alopecia**

Alopecia

 

Alopecia

 

Alopecia

 

Alopecia

Tris(2-hydroxyethyl) isocyanurate triacrylate 50% (w/v) in DMF

2772

 

2798

 

2791

 

2766

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

Tris(2-hydroxyethyl) isocyanurate triacrylate 25% (w/v) in DMF

2796

 

2792

 

2771

 

2794

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

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

2762

 

2769

 

2793

 

2800

 

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

Notes:

1. BT: before treatment; AT: after treatment

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

 

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 1 (skin sensitising) based on GHS criteria
Conclusions:
In conclusion, under the conditions of the present assay, Tris(2-hydroxyethyl) isocyanurate triacrylate, tested in a suitable vehicle, was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.
Executive summary:

The aim of this study was to determine the skin sensitisation potential of Tris(2-hydroxyethyl) isocyanurate triacrylate 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 N,N-dimethylformamide (abbreviated as DMF). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100 % (w/v).

 

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

 

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

- three groups received Tris(2-hydroxyethyl) isocyanurate triacrylate (formulated in DMF) at 100, 50 and 25 % (w/v) concentrations,

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

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

 

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. At the 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 during the study. Alopecia around the ears was observed in the 100 % (w/v) dose group on Days 3-6 or Days 4-6. Test item precipitate or minimal amount of test item precipitate was observed on the ears of the animals in all dose groups.

 

The stimulation index values were 30.4, 21.2 and 24.2 at concentrations of 100, 50 and 25 % (w/v), respectively. The test item gave a relatively strong positive response without a clear reduction at the lowest dose level; hence the obtained data did not allow the calculation of the EC3 value of the test item. Therefore, no sub-category according to GHS system can be determined for the test item.

 

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, Tris(2-hydroxyethyl) isocyanurate triacrylate, tested in a suitable vehicle, was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

The following classification/labelling is triggered:

Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 6) 2015: Category 1 without subcategory

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Skin sensitisation in mice (Valiczko 2016):

The aim of this study was to determine the skin sensitisation potential of Tris(2-hydroxyethyl) isocyanurate triacrylate 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.

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

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each: three groups received Tris(2-hydroxyethyl) isocyanurate triacrylate (formulated in DMF) at 100, 50 and 25 % (w/v) concentrations, the negative control group received the vehicle (DMF), the positive control group received 25 % (w/v) HCA (dissolved in DMF).

The test item solutions were applied on the d*orsal 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. At the 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 during the study. Alopecia around the ears was observed in the 100 % (w/v) dose group on Days 3-6 or Days 4-6. Test item precipitate or minimal amount of test item precipitate was observed on the ears of the animals in all dose groups.

The stimulation index values were 30.4, 21.2 and 24.2 at concentrations of 100, 50 and 25 % (w/v), respectively. The test item gave a relatively strong positive response without a clear reduction at the lowest dose level; hence the obtained data did not allow the calculation of the EC3 value of the test item. T

In conclusion, under the conditions of the present assay, Tris(2-hydroxyethyl) isocyanurate triacrylate, tested in a suitable vehicle, was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Justification for classification or non-classification

 Based on the in vivo study, Tris(2-hydroxyethyl) isocyanurate triacrylate should be classified as Skin sens.1 (H317) without subcategory according to the Regulation EC no.1272/2008.

Justification : The test item gave a relatively strong positive response without a clear reduction at the lowest dose level; hence the obtained data did not allow the calculation of the EC3 value of the test item. Therefore, no sub-category according to CLP can be determined for the test item.