Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

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:
2010-02-01 to 2010-03-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Temporary deviations from the minimum levels of the relative humidity. Historical laboratory data do not indicate an effect of the deviations.
GLP compliance:
yes
Remarks:
Quality assurance statement and inspection report by NOTOX Quality Assurance Unit.
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxy-3-{4-[2-(4-{2-hydroxy-3-[(2-methylprop-2-enoyl)oxy]propoxy}phenyl)propan-2-yl]phenoxy}propyl 2-methylprop-2-enoate
EC Number:
701-308-4
Cas Number:
36425-15-7
Molecular formula:
n/a
IUPAC Name:
2-hydroxy-3-{4-[2-(4-{2-hydroxy-3-[(2-methylprop-2-enoyl)oxy]propoxy}phenyl)propan-2-yl]phenoxy}propyl 2-methylprop-2-enoate
Details on test material:
- Name of test material (as cited in study report): Substance 2, NLP 500-089-0
- Molecular formula: C29H36O8
- Molecular weight: Mn= approx. 700
Mw= approx. 820
(GPC, relative to polystyrene)
- Substance description: Clear light yellow-greenish to light brown very high viscous liquid
- Physical state: Liquid

- Analytical composition and purity:
Approx. 80% BisGMA
Approx. 13% Polymeric methacrylates (more Bisphenol-As and glycidyl in chains)

- Purity test date: Not given, analysis data provided by sponsor.
- Batch No.: 7007783
- Expiration date of the Batch: January 13, 2011 (allocated by NOTOX, 1 year after receipt of the test substance).
- Storage condition of test material: In refridgerator (2-8°C) protected from light.

- Stability under test conditions:
Solubility in acetone: Not indicated
Solubility in Olive oil: Not indicated
Stability in acetone: Yes
Stability in Olive oil: Yes, long.
Not stable if exposed to direct sunlight for days/weeks
Stable at temperatures up to 80°C for max. 1-2 hours

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, LArbresle Cedex, France
- Mouse strain: CBA/J, inbred, SPF-Quality
- Age at study initiation: approx. 9 weeks
- Number of animals: 20 females (nulliparous and non-pregnant) (4 groups with 5 in each)
- Weight at study initiation: Average weight: 20.95 g; Range from 19-23 g. Bodyweight variation was within ± 20% of the sex mean.
- Housing: Individual housing in Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son, Wonham Mill Ltd, Surrey United Kingdom) was supplied as cage enrichment. The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum.
- Water (e.g. ad libitum): Fresh tapwater ad libitum
- Acclimation period: Acclimatization for at least 5 days before start of the treatment under laboratory conditions, accommodated as described except that the animals were group house in Macrolon cages (MIII type, Height 18 cm).
- Identification: Tail mark with marker pen
- Health inspection: Health inspection performed prior to treatment to ensure that animals were in a good state of health. Special attention was paid to the ears which were intact and free of any abnormalities.
- Reliability check: The results of the reliability tests with Hexylcinnamaldehyde performed not more than 6 months previously, were summarized in an appendix to the report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 °C (actual range: 18.5-22.7°C)
- Humidity (%): 40-70 % (actual range: 39-65%)
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 h artificial fluorescent light and 12 h dark per day.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
Choice based on trials performed at Notox
Concentration:
0, 10, 25, and 50% (w/w) test substance in acetone/olive oil (4:1 v/v).
No. of animals per dose:
5
Details on study design:
TEST SUBSTANCE FORMULATION
- Preparation of test substance formulations: The test substance solutions were prepared within 4 hours prior to each treatment. Dilutions were made w/w and are given in %. No adjustments were made for the specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels. In order to obtain homogeneity, the test substance formulations were heated in a water bath with a max. temperature of 50°C for max. 1 h and 10 min. the test substance formulations were allowed to cool down to a temperature of max. 40°C before dosing.
- Rationale for choice of vehicle: The vehicle was selected on the basis of trial formulations performed at NOTOX and on the test substance data supplied by the sponsor.
-
Preliminary irritation study/ RANGE FINDING TEST:
- Compound solubility: Homogeneity achieved as judged by visual inspection.
- Concentrations: 25% and 50% w/w test substance.
- Method: 2 young adult CBA/J mice one for each concentration was treated by application of a test formulation for 3 consecutive days. 3-4 h after the last exposure, the irritation of the ears was assessed. Body weights were determined on day 3 and the animals were sacrificed after the final observation and no necropsy performed.
- Irritation: On day 3 slight erythema (score 1) and no oedema (score 0) was observed on both ears at the two mice treated with 25% and 50% test substance, respectively.
- Lymph node proliferation response: Was not investigated.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA; Epidermal exposure to 25 µl test substance per ear for 3 consecutive days (induction) followed by evaluation of lymphatic node cell proliferation in the regional lymph nodes using 3H-methyl thymidine incorporation on day 6 after first exposure.
- Criteria used to consider a positive response:
Toxicity and Mortality
Body weights
Irritation:
Erythema: No erythema: 0; Slight erythema: 1;Well-defined erythema: 2;Severe erythema: 3
Oedema: No oedema: 0; Slight oedema: 1;Moderate oedema: 2; Severe oedema: 3
Macroscopy of the auricular lymph nodes and surrounding area.
Radioactivity measurements in lymph node cells collected after 3H-methyl thymidine incorporation (for labeling of proliferating cells) on day 6. Used for calculation of the Stimulation Index (SI). The SI is the ration of the DMP/group compared to the DMP/vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION:
- No. of exposures: 3
- Exposure period: 3 consecutive days
- Test groups and concentrations: 3 with five animals in each group: 10%, 25% and 50% test substance in acetone/olive oil
- Control group: 0% test substance in acetone/olive oil
- Site: epidermal application on the ears
- Frequency of applications: once a day at approx. same time of day.
- Duration: 3 day exposure period and 3 day follow up period. Injection with 3H-methyl thymidine through the tail vein on day 6.
- Evaluation (hr after challenge):
Toxicity and Mortality: Twice daily
Body weight: On day 1 and 6
Irritation: On day 3 3-4 hours after last exposure.
Necropsy including macroscopy of the auricular lymph nodes: On day 6.
Radioactivity measurements: Lymp nodes collected on day 6

Method for measurement of Lymph Node Cells (LNC) proliferation
- Radioactive labeling of proliferating LNCs: Each animal was injected via the tail vein with 0.25 ml of sterile phosphate buffered saline (PBS; Merck, Darmstadt, Germany) containing 20 µCi of 3H-methyl thymidine (Perkin Elmer Life and Analytical Sciences, Boston, MA, USA).
After approx. 5 h all animals were killed by i.p. injection with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The auricular lymph node of each ear was excised and the relative size of the nodes was estimated by visual inspection. Any abnormalities and surrounding area were recorded. The nodes were pooled for each animal in 3 ml of PBS.
- Tissue processing for radioactive LNCs: Single cell suspension of LNC was prepared in PBS by gentle separation through a stainless steel gauze (diameter 125 µm). LNC were washed twice with excess of PBS, and centrifuged at 200 g for 10 min at 4°C. To precipitate DNA, the LNC were exposed to 5% trichloroacetic acid (TCA; Merck, Darmstadt, Germany) and stored in the refrigerator until next day. On day 7 the precipitates were recovered by centrifugation, resuspended in 1 ml TCA and transferred to 10 ml of Ultima Gold Cocktail-scintillation fluid (PerkinElmer Life and Analytical Sciences, Boston, MA, USA). Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ±0.2% or a maximum of 5 minutes, whichever came first. The scintillation counter was programmed to automatically subtract background and convert counts per minute (CPM) to disintegrations per minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistical analyses were performed. A stimulation index of 3, indicative of LNC proliferation was regarded the criterion for categorizing the test substance as a skin irritant.

Results and discussion

Positive control results:
Reliability test performed using alpha-hexylcinnamaldehyde in acetone/olive oil vehicle. study conducted in sept./october 2009 in 11 weeks old female CBA/J mice as described for the main study.
Results:
Group % Test substance in Acetone/Olive oil
(4:1 v/v) Mean DPM ± SEM SI ± SEM
1 0 % 601± 74 1.0 ± 0.2
2 5% 856 ± 178 1.4 ± 0.3
3 10% 692 ± 129 1.2 ± 0.3
4 25% 3070 ± 479 5.1 ± 1.0

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Variability:
+/- 0.52
Test group / Remarks:
0 (Control)
Key result
Parameter:
SI
Value:
0.9
Variability:
+/- 0.4
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
0.8
Variability:
+/- 0.3
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.7
Variability:
+/- 0.7
Test group / Remarks:
50%

Any other information on results incl. tables

Main study:

Mortality and toxicity: No mortality occurred and no signs of systemic toxicity were observed in any of the animals.

Skin reactions: The slight irritation of the ears as shown by all animals treated with 10 - 50% of the test substance was considered not to have a toxicological significance on the activity of the lymph nodes. No oedema was observed in any of the animals (see table below).

Macroscopic examination of the auricular lymph nodes and surrounding area: All auricular lymph nodes of the animals in the experimental and control groups were evaluated normal in size. No macroscopic abnormalities in the surrounding tissue were noted in any animal.

Body weight: Body weights and body weight gains of the experimental animals remained in the same range as the control animals over the study period. The slight body weight loss noted in some animals was considered not toxicologically significant.

Table 2: Skin reactions, body weights and relative size of auricular lymph nodes

N: size of auricular lymph node considered normal.

Group

Test sub-stance

Animal

Day 1

Day 3

Skin reactions

Day 6

 

 

Body weight

left

right

Body weight

Node sizes

 

% w/w

No.

g

Eryth.

Oed.

Eryth.

Oed.

g

left

right

1

0%

1

21

0

0

0

0

21

N

N

 

 

2

23

0

0

0

0

22

N

N

 

 

3

21

0

0

0

0

21

N

N

 

 

4

23

0

0

0

0

22

N

N

 

 

5

23

0

0

0

0

22

N

N

2

10%

6

22

1

0

1

0

22

N

N

 

 

7

19

1

0

1

0

19

N

N

 

 

8

22

1

0

1

0

20

N

N

 

 

9

20

1

0

1

0

19

N

N

 

 

10

19

1

0

1

0

18

N

N

3

25%

11

22

1

0

1

0

22

N

N

 

 

12

20

1

0

1

0

19

N

N

 

 

13

21

1

0

1

0

21

N

N

 

 

14

19

1

0

1

0

19

N

N

 

 

15

23

1

0

1

0

24

N

N

4

50%

16

20

1

0

1

0

18

N

N

 

 

17

19

1

0

1

0

18

N

N

 

 

18

19

1

0

1

0

20

N

N

 

 

19

23

1

0

1

0

22

N

N

 

 

20

20

1

0

1

0

20

N

N

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Conclusions:
Small Vinyl Ester cannot be regarded a skin sensitizer, as evaluated by the LLNA test in mouse and recommendations made in the OECD test guideline 429. The calculated SI values for substance concentrations of 10, 25, and 50% w/w in acetone/olive oil (4:1 v/v) were 0.9, 0.8, and 1.7, respectively. Reliability of the LLNA test method as performed by NOTOX was checked with Hexylcinnamaldehyde (conducted every 6 months).
Executive summary:

The ability of Small Vinyl Ester to induce contact hypersensitivity was tried experimentally using the Local Lymph Node Assay in mice according to the OECD Test Guideline 429.

The report covers data and results from a preliminary irritation study, determining the highest test substance concentration to be used in the main study, and the main study. The test substance was diluted in a vehicle of acetone and olive oil (4:1 v/v) in concentrations of 10, 25, and 50%. For control vehicle alone was used.Reliability of the LLNA test method as performed by NOTOX was checked with Hexylcinnamaldehyde (conducted every 6 months), and results from the latest reliability test was reported in an appendix to the report.

In the main study, 9 weeks old female CBA/J-mice were used, randomized into 4 groups with 5 in each group. Three test groups and 1 control group. The mice were exposed to the test substance by epidermal application of 25 µl of test suspension to each ear for 3 consecutive days. Macroscopic signs of skin irritation was scored 3-4 hours after the last application. Injection with 3H-methyl thymidine for labelling of proliferating lymph node cells was performed on day 6, and after 5 hours the animals were killed, the auricular lymph nodes collected and processed for collection of cellular DNA. Cell proliferation stimulation, presented as the Stimulation Index (SI) was calculated from the number of Disintegrations Per Minute in the lymph node cell DNA samples collected from animals in each group.

The SI values for substance concentrations of 10, 25, and 50% w/w in acetone/olive oil (4:1 v/v) were 0.9, 0.8, and 1.7, respectively. This is below the limit of SI = 3 for categorization as a skin irritant, recommended by the test guideline. Hence, the test substance cannot be regarded a skin irritant.