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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:
26 September 2016 - 8 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 22, 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Regulation (EC) No. 440/2008
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Test material form:
other: liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
mouse
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation:
- Weight at study initiation:8 to 10 weeks old
- Housing: suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet: ad libitum
- Water: ad libitum
- 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:
0% (vehicle control), 10%, 30% and 100% (test item)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
A preliminary screening test with one animal is performed with undiluted test material observed for local skin Irritation, clinal oberservations and ear thickness measurements were done.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
Selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card.

TREATMENT PREPARATION AND ADMINISTRATION:
Test item was used undiluted, and freshly prepared as a solution (10, 30% v/v/) in acetone/olive oil 4:1. Four groups of five mice were treated with 25 µL per ear 0, 10, 30 and 100% v/v concentrations in acetone/olive oil 4;1 of testing material for 3 consecutive days (Days 1, 2, 3). On day 6, all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd): a total of 20 µCi to each mouse. All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination). At termination, for each individual the draining auricular lymph nodes were excised and processed, and a single cell suspension was prepared (individual animal approach). The radioactive disintegrations per minute per lymph node and the stimulation index are determined using the Beckman LS6500 scintillation system.

EVALUATION
The disintegrations per minute (dpm) value was determined for each test group. This information was used to calculate the Stimulation Index (SI) for each of the test groups (disintegrations per minute of treatment group / disintegrations per minute of control group). A positive response is indicated when one or more test groups shows a SI of 3. The EC3 value is the concentration at which a 3-fold increase of
lymph node proliferation is observed. Interpolation is used to determine the EC3 value between two test concentrations
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Individual and group mean disintegrations per minute values were assessed for dose response relationships.

Results and discussion

Positive control results:
A current positive control study was performed (study number SK71FG, 8 April 2016 to 14 April 2016). Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at concentrations of 5%, 10% or 25% (v/v). A further group of five animals was treated with acetone/olive oil 4:1 alone and served as the vehicle control group. The concentration of α-Hexylcinnamaldehyde, tech., 85% expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 20%. α-Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Test group / Remarks:
All test groups
Remarks on result:
not determinable
Remarks:
All SI < 3
Parameter:
SI
Value:
0.85
Test group / Remarks:
10% (v/v)
Parameter:
SI
Value:
1.14
Test group / Remarks:
30% (v/v)
Parameter:
SI
Value:
1.12
Test group / Remarks:
100% (v/v)
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean DPM with standard deviation:
- Vehicle control group: 3862.88 (±2115.84)
- 10% (v/v): 3282.69 (±1089.26)
- 30% (v/v): 4406.17 (±730.15)
- 100% (v/v): 4335.68 (±1474.10)

DETAILS ON STIMULATION INDEX CALCULATION
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.
- 10% (v/v): 0.85
- 30% (v/v): 1.14
- 100% (v/v): 1.12

EC3 CALCULATION
The test item was considered to be a non-sensitizer under the conditions of the test.

CLINICAL OBSERVATIONS
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

In the preliminary screening test there were no signs of systemic toxicity or visual local skin irritation noted. No irritation was indicated by an equal to or greater than 25% increase in mean ear thickness. Based on this information the undiluted test item and the test item at concentrations of 30% and 10% v/vinacetone/olive oil 4:1 were selected for the main test.

Applicant's summary and conclusion

Interpretation of results:
other: not classified
Remarks:
based on CLP criteria
Conclusions:
Under the conditions of this test, the test substance did not induce a Stimulation Index (SI) above 3 and therefore an EC3 value could not be calculated. Based on these results, the substance is considered not to be a sensitiser and does not need to be classified for skin sensitisation in accordance with the criteria outlined in Annex I of 1272/2008/EC (CLP).
Executive summary:

The Local Lymph Node Assay (according to OECD 429) was conducted to determine the sensitising potential of Muguesia in mice. Lymph node proliferation was determined after exposure to 0%, 10%, 30% or 100% test substance in vehicle (Acetone/Olive oil (4:1 v/v)), using radioactivity counts (DPM). A pooled approach (per test group) was used. Stimulation indices were calculated. Measured disintegrations per node/minute (pooled) were 3863, 3283, 4406 and 4336 for the 0%, 10%, 30% and 100% dosing groups, respectively. Corresponding stimulation indices (SI) were calculated to be 0.85, 1.14 and 1.12 for the 10%, 30% and 100% concentration, respectively. Under the conditions of this test, the test substance did not induce a Stimulation Index (SI) above 3 and therefore an EC3 value could not be calculated. Based on these results, the substance is considered not to be a sensitiser and does not need to be classified for skin sensitisation in accordance with the criteria outlined in Annex I of 1272/2008/EC (CLP).