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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:
25 May - 26 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methoxyethyl acrylate
EC Number:
221-499-3
EC Name:
2-methoxyethyl acrylate
Cas Number:
3121-61-7
Molecular formula:
C6H10O3
IUPAC Name:
2-methoxyethyl acrylate
Details on test material:
- Name of test material (as cited in study report): 2-methoxyethyl acrylate
- Physical state: clear colourless liquid
- Analytical purity: 99.9%
- Lot/batch No.: C220424SP6
- Expiration date of the lot/batch: 2013-05-31
- Storage condition of test material: room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: the animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood wood flakes.
- Diet: 2014C Teklad Global Rodent diet (Harlan Laboratories UK Ltd., Oxon, UK)
- Water: tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25 and 50% (v/v), 100% (undiluted)
No. of animals per dose:
preliminary screening test: 1
main assay: 4
Details on study design:
RANGE FINDING TESTS: in a preliminary screening test, the systemic toxicity/irritancy potential of the test item was assessed in each one mouse treated once daily with 25 µL of the undiluted test item (100%) and the test item at concentrations of 50%, 25% or 10% v/v in acetone/olive oil 4:1 on the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily and any clinical signs of toxicity, if present, were also recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post dose on Day 3 and on Day 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

- Compound solubility: the test substance at 50% (v/v) in acetone/olive oil 4:1 was well soluble, and thus suitable for dosing.
- Irritation: no irritation indicated by an equal to or greater than 25% increase in mean ear thickness was noted. Very slight erythema was noted in animals treated with the test item at concentrations of 25% and 10% v/v in acetone/olive oil 4:1 during Days 3-5. No visual local skin irritation was noted in animals treated with the undiluted test item or the test item at a concentration of 50% v/v in acetone/olive oil 4:1.
- Clinical signs: no signs of systemic toxicity were observed.
Based on this information, the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³H-methyl thymidine (³HTdR) incorporation determined by β-scintillation
- Criteria used to consider a positive response: the test item will be regarded as a sensitiser if at least one concentration of the test item results in a 3-fold or greater increase in ³HTdR incorporation compared to control values. Any test item failing to produce a 3-fold or greater increase in ³HTdR incorporation will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION: based on the preliminary screening test, groups of 4 mice were treated with the undiluted test item (100%) or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The test substance (25 µL) was daily applied to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3) using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner. Five days following the first topical application of the test item or vehicle (on Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing ³HTdR at 80 µCi/mL (specific activity 2.0 Ci/mmoL, ARC UK Ltd), giving a total of 20 µCi to each mouse. Five hours later, draining auricular lymph nodes were excised and pooled for each experimental group. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through 200-mesh stainless steel gauze. The lymph node cells per group were each rinsed through the gauze with 4 mL of PBS into a petri dish and then transferred to the respective centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted by centrifugation and the obtained pellet was resuspended in 10 mL of PBS and re-pelleted. Then, the pellet was resuspended in 3 mL of 5% trichloroacetic acid (TCA) and precipitated at 4 °C for a period of approx. 18 h. After centrifugation, the pellet was resuspended in 5% TCA and ³HTdR incorporation was measured by β-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean ear thickness was determined in control and treated animals.

Results and discussion

Positive control results:
The current positive control substance α-hexylcinnamaldehyde at 25% v/v in acetone/olive oil 4:1 produced a stimulation index (SI) of 5.76, thus fulfilling the reliability criteria for the LLNA (SI > 3).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
9.2
Test group / Remarks:
25% in AOO 4:1
Key result
Parameter:
SI
Value:
12.84
Test group / Remarks:
50% in AOO 4:1
Key result
Parameter:
SI
Value:
11.55
Test group / Remarks:
100%

Any other information on results incl. tables

Table 1. Results of the LLNA

Concentration (% v/v) in acetone/olive oil 4:1

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

12918.23

1614.78

na

na

25

11883.60

14860.45

9.20

positive

50

165885.80

20735.73

12.84

positive

100

149168.70

18646.09

11.55

positive

dpm = Disintegrations per minute

a = Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b = Stimulation Index of 3.0 or greater indicates a positive result

na = Not applicable

CLINICAL OBSERVATIONS

No mortalities and no signs of systemic toxicity were observed during the study.

 

BODYWEIGHTS

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
CLP: Skin Sens. 1, H317