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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:
The study was conducted between 15 June 2009 and 15 July 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, ME 04609
- Age at study initiation: 8 weeks
- Weight at study initiation: 19 to 25 g
- Housing: 5 animals/cage (randomly allocated)
- Diet (e.g. ad libitum): ad libitum (Harlan Teklad Certified Rodent Chow 7012C)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 44-62
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: ethanol/diethyl phthalate 1:3
Concentration:
2.5 %, 5%, 10%, 25% or 50% v/v in EtOH/DEP 1:3
No. of animals per dose:
5
Details on study design:
MAIN TEST

Test Item Administration
Groups of five mice were treated with the test item at concentrations of 2.5 %, 5%, 10%, 25% or 50 % v/v in EtOH/DEP. No justification on the choice of vehicle is provided. The doses were chosen on the basis of avoiding systemic toxicity and overtly induced skin irritation, according to reported uses of the test material. The mice were treated by daily application of 25 µl of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.
The positive control animals were similarly treated to the test animals except that 25 µl of the positive control item, α Hexylcinnamaldehyde, at a concentration of 5%, 15% and 35 % v/v in ethanol/diethyl phthalate 1:3 was applied to the dorsal surface of each ear.

3H-Methyl Thymidine Administration
On day 6, all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR:80µCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed before and after dosing, 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 animals were also examined daily for erythema and edema on the site of application.

Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. .

Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared. The lymph node cells were rinsed with PBS and precipitated with 5% trichloroacetic acid during night. The lymph node cells suspension was transferred to a centrifuge tube and thereafter, the pellets were resuspended in 1 ml TCA and transferred to scintillation fluid vials for the measurement of the radioactive material.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Increases in the radioactivity compared to the vehicle control were recorded. Individual DPM values were analyzed with log values. Dunett's test was used to determine significance when required. The EC3 was calculated as follows:

EC3=c+[(3-d)/(b-d)](a-c),

data points between the SI=3 lie in the coordinates (a,b) and (c,d)

Results and discussion

Positive control results:
The positive control item, α-Hexylcinnamaldehyde, gave a Stimulation Index of greater than 3 when tested at a concentration of 35% v/v.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.7
Test group / Remarks:
2.5% test substance
Remarks on result:
other: 2.5%
Key result
Parameter:
EC3
Remarks:
%
Value:
19.2
Key result
Parameter:
other: NOEC
Remarks:
%
Remarks on result:
other: 10%
Parameter:
SI
Value:
1.5
Test group / Remarks:
5% test substance
Remarks on result:
other: 5%
Parameter:
SI
Value:
1.9
Test group / Remarks:
10% test substance
Remarks on result:
other: 10%
Parameter:
SI
Value:
3.7
Test group / Remarks:
25% test substance
Remarks on result:
other: 25%
Parameter:
SI
Value:
8.1
Test group / Remarks:
50% test substance
Remarks on result:
other: 50%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean DPM at 0 (vehicle), 2.5, 5, 10, 25, and 50% were 201.6, 348.5, 311.4, 387.0, 745.1 and 1629.8, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index is calculated by dividing the test group DPM through the vehicle group DPM.

EC3 CALCULATION
The EC3 was calculated using the formula: EC3 = c+[(3-d/(b-d)](a-c), where the dta points lying immediately above and below the SI value of 3 have the coordinates (a, b) and (c, d).

CLINICAL OBSERVATIONS
No mortality occurred. No erythema and edema was observed. Wet ears were observed in some test groups on different days. The lymph nodes of the highest dose group appeared enlarged compared to controls. There were no other findings.

BODY WEIGHTS
Mean body weights and body weight changes showed no statistical significant differences between the vehicle and test groups.

Applicant's summary and conclusion

Interpretation of results:
other: Skin Sensitiser 1B
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
The test item was considered to be a sensitiser under the conditions of the test and resulted in an EC3 of 19.2 and a NOEC of 10%. Based on this value, the substance can be considered a skin sensitiser.
Executive summary:

The skin sensitisation potential of the test substance has been tested according to the OECD TG 429 (Local Lymph Node Assay) guideline. At 2.5, 5, 10, 25 and 50% the substance showed SI values of 1.7, 1.5, 1.9, 3.7 and 8.1, respectively. No signs of irritation or other toxicity were detected. The EC3 was calculated to be 19.2% and the NOEC is 10%. The substance was found to be a skin sensitiser 1B under the conditions of this test.