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Administrative data

Description of key information

Skin sensitisation (OECD TG 429): Sensitising (EC3 of 19.2%)

Respiratory sensitisation: Not sensitising in absence of human data and absence of structural alerts for respiratory sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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
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)
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
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)
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.
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.
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin sensitisation in the LLNA

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 10%. The substance was found to be a skin sensitiser under the conditions of this test.

Skin sensitisation in HRIPT tests supporting information not used for DNEL derivation

In addition, a human Repeated Insult Patch Test (hRIPT) is available in which 41 volunteers exposed to 2.5% test substance in alcohol SDA 39. 0.5mL sample was applied to the skin with a 1 x 1 inch patch. The patch was removed 24 h after application. The patch was applied on the same area each time during the 9 inductions. For the final challenge duplicate patches were applied, one on the original test site and one on a skin site that did not previously receive test patches. 41 panellists showed little or no primary irritation. There was no evidence of sensitisation (IFF 1971).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The substance is not a respiratory sensitiser in absence of human data indicating such effects. In addition, the respiratory sensitisation is assessed using the integrated evaluation strategy for respiratory sensitisation data in the ECHA guidance (R7A, Fig. 7.3-2, 2014).

1)     The substance is a skin sensitiser;

2)     The substance does not belong to the di-isocyanates;

3)     the substance has no structural alerts or is structurally related to chemicals causing respiratory sensitisation as presented in Table R.7.3-1 in the ECHA guidance of 2008 or those provided in the following document: http://ec.europa.eu/health/scientific_committees/docs/annex6_respiratory.pdf

Therefore the substance is not considered to be a respiratory sensitiser.

Justification for classification or non-classification

Based on the available information, the substance should be classified as sensitising to the skin in accordance with the criteria outlined in the EU CLP Regulation (1272/2008/EC and its amendments) resulting in Skin Sens. 1B / H317: May cause an allergic skin reaction.

In absence human data indicating respiratory sensitisation and using the ITS in the ECHA guidance (R.7a, 2014) the substance is not considered to be a respiratory sensitiser in accordance with the criteria outlined in the EU CLP (EC 1272/2008 and its amendments)