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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11.06.2016 - 01.07.2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name of test material (as cited in study report): 1,6-Hexanediol dimethacrylate;

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- CBA/CaOlaHsd mice, nulliparous, non-pregnant
- Age at study initiation: 10-11 weeks (at beginning of treatment)
- Weight at study initiation: 20.7 g ± 1.1 g
- Housing: single; Makrolon TypeIII, with wire mesh top
- Diet (e.g. ad libitum): 2018C Tekland Global 18% protein rodent diet (certified),ad libitumpelleted standard, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 5 days prior to start of dosing under test conditions after health examination.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 45-65 %
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2007-02-07 To: 2007-02-13

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
-non-GLP pretest (test substance): 50%; 100%
-Main study (test substance): 10%, 25%; 50%
-Positive control: α-Hexylcinnamaldehyde in acetone: olive oil (4+1)
-Negative control: vehicle
No. of animals per dose:
5 female per dose
Details on study design:
RANGE FINDING TESTS: Concentrations were tested on two mice on one ear each. Test concentrations: 50 and 100 % in acetone/olive oil 4.1 v/v The animal treated with 50% test item concentration showed an erythema of the ear skin (score 1 on day 5; and score 2 on days 3 and 4). The animal treated with 100% test item concentration showed an erythema of the ear skin (score 1 on day 6; and score 2 between day 3 and 5). Furthermore, the animal treated with 100% test item concentration showed unspecific signs of systemic toxicity (slight dehydration and body weight loss on days 3 and 4). Thus, the test item in the main study was assayed at 10, 25 and 50%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

Experimental Design and Procedures Topical application Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10, 25 and 50% in acetone/olive oil (4+1, v/v). The application volume, 25μL/ear/day, was spread over the entire dorsal surface (∅ ∼8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of3H-methyl-thymidine
Five days after the first topical application (day 6) 250μL of phosphate-buffered saline containing 20.2μCi of3H-methyl thymidine (equivalent to 80.8μCi/mL3HTdR) were injected into each test and control mouse via the tail vein.

Determination of incorporated3HTdR
Approximately five hours after treatment with3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death. The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200μm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in aβ-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

Interpretation of raw data
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled: First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index. Second, that 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.
Observations
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period: Mortality / Viability: At least once daily from experimental start to necropsy. Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with3HTdR. Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6). Ear weights: In the pre-test after sacrifice; biopsy punches were taken from each ear.

Clinical signs (local / systemic): Clinical signs (local irritation at the application site or
systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.
 
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation). The Dean-Dixon-Test was used for identification of possible outliers (performed with Microsoft
Excel 2007). However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
A positive control performed with alpha-Hexylcinnamaldehyd in April 2014 resulted in an S.I. of 2.51 at 5% (w/v) alpha-Hexylcinnamaldehyd, 1.76 at 10% and 6.79 at 25%. An EC3 of 13.% (w/v) was calculated.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.88
Test group / Remarks:
10% in acetone/olive oil (4+1, v/v)
Remarks on result:
other: DPM/lymph node: 2145.4
Parameter:
SI
Value:
2.24
Test group / Remarks:
15% in acetone/olive oil (4+1; v/v)
Remarks on result:
other: DPM/lymph node: 2556.0
Parameter:
SI
Value:
2.87
Test group / Remarks:
50% in acetone/ olive oil (4+1, v/v)
Remarks on result:
other: DPM/lymph node: 3268.6

Any other information on results incl. tables

In this study Stimulation Indices (S.I.) of 1.88, 2.24 and 2.87 were determined with the test item at concentrations of 10, 25 and 50% in acetone/olive oil (4+1, v/v), respectively.

The test item 1,6-Hexanediol dimethacrylate was not a skin sensitiser under the test conditions of this study.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this Local Lymphnode Assay 1,6-Hexanediol dimethacrylate was not a dermal sensitizer.
Executive summary:

In a dermal sensitisation study according to OECD Guideline 429 (adopted 22. July 2010) with 1,6 Hexanediol dimethacrylate in acetone:olive oil (4+1, v/v), groups of 5 female CBA/CaOlaHsd mice were tested using the LLNA method.

STIMULATION INDICES (S.I.) of 1.88, 2.24 and 2.87 were determined with the test substance at concentrations of 10%, 25% and 50% (w/v) in acetone:olive oil (4+1, v/v), respectively. The positive control substance wasα-Hexylcinnamaldehyde, which gave an EC3 at 13.7.1% (w/v).

A result is regarded as positive when the S.I. is ≥3.

Based on these criteria, the test substance was found not to be a sensitiser at any concentration tested. In this study, 1,6-Hexanediol dimethacrylate is not a dermal sensitiser.

 

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