Registration Dossier

Administrative data

Description of key information

The test material is a skin sensitiser in accordance with EU criteria.

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:
27 April 2005 to 03 May 2005
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)
Deviations:
no
Principles of method if other than guideline:
Animal number 59 in group 4 died during thymidine dosing and was hence excluded from the study. The integrity of the study is not affected by the loss of one animal in a group.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/Ca
- Age at study initiation: Young adults (8-12 weeks of age)
- Weight at study initiation: 16.4 to 20.1 g (study day 1)
- Housing: A maximum of 4 mice was housed per cage, in cages suitable for animals of this strain and weight range. Environmental enrichment provided included tents, bases and nestlets.
- Diet: ad libitum
- Water: ad libitum mains water supplied by an automatic system
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 to 70 %
- Air changes: A minimum of 15 changes per hour
- Photoperiod: Artificial light with a cycle of 12 hours of light/12 hours of darkness
Vehicle:
other: 1:3 Ethanol/Diethylphthalate (1:3 EtOH:DEP)
Concentration:
2.5, 5, 10, 25 and 50 % w/v
No. of animals per dose:
4 females per dose
Details on study design:
MAIN STUDY
Approximately 25 µL of the appropriate preparation of the test material in 1:3 EtOH:DEP was applied, using a variable volume micro-pipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using 1:3 EtOH:DEP alone. The procedure was repeated daily for 3 consecutive days.
Three days after the third application, all the animals were injected, via the tail vein, with approximately 250 µL of phosphate buffered saline (PBS) containing 20 µCi of a 2.0 Ci/mmol specific activity ³H-methyl thymidine. Approximately 5 hours later, the animals were humanely killed by inhalation of halothane vapour followed by cervical dislocation. The draining auricular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.
A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed three times by centrifugation with approximately 10 mL of PBS. Approximately 3 mL of 5 % w/v trichloroacetic acid (TCA) was added and, after overnight precipitation at 4 °C, the samples were pelleted by centrifugation and the supernatant was discarded. The cells were then re-suspended in approximately 1 mL of TCA.
The lymph node suspensions were transferred to scintillation vials and 10 mL of scintillant (Optiphase) was added prior to β-scintillation counting using a Packard Tri-Carb 3100TR Liquid Scintillation Counter.

-Clinical observations and body weights
Animals were checked at least once daily for signs of systemic toxicity and the bodyweight of each animal was recorded prior to dosing on day 1 and prior to injection of ³H-methyl thymidine on day 6.

TREATMENT PREPARATION AND ADMINISTRATION
- Dose preparations: All dose preparations were used within 24 hours of preparation. No correction was made for the purity of the active ingredient in the test material. Stability and achieved concentration were not determined.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The application of hexylcinnamaldehyde at concentrations of 5, 10 and 25 % w/v in acetone:olive oil (4:1) resulted in a greater than 3-fold increase in isotope incorporation at the 25 % concentration. Therefore, hexylcinnamaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.
Parameter:
SI
Value:
1
Test group / Remarks:
2.5%
Parameter:
SI
Value:
2.7
Test group / Remarks:
5%
Parameter:
SI
Value:
2.4
Test group / Remarks:
10%
Parameter:
SI
Value:
3.8
Test group / Remarks:
25%
Parameter:
SI
Value:
8.3
Test group / Remarks:
50%
Key result
Parameter:
EC3
Remarks:
test item concentration in %
Value:
16.4

Table 1: Skin Sensitisation Potential

Concentration (% w/v)

Number of Lymph Nodes Assayed

Disintegrations per Minute (DPM)

DPM per Lymph Node

Test:Control Ratio

0 (Vehicle)

8

6458

807

N/A

2.5

8

6518

815

1.0

5

8

17 482

2185

2.7

10

8

15 285

1911

2.4

25

6

18 159

3027

3.8

50

8

53 752

6719

8.3

HCA Vehicle

8

4397

550

N/A

HCA 5

8

6402

800

1.5

HCA 10

8

9771

1221

2.2

HCA 25

8

28921

3615

6.6

N/A = Not applicable

HCA = hexylcinnamaldehyde (positive control)

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the study, the test material was considered to be a skin sensitiser.
Executive summary:

A Local Lymph Node Assay study in the mouse was conducted to investigate the skin sensitisation potential of the test material in accordance with the standardised guidelines OECD 429 and US EPA OPPTS 870.2600 under GLP conditions.

The test material was applied to the dorsal surface of each ear of female CBA/Ca mice at concentrations of 2.5, 5, 10, 25 and 50 % w/v in 1:3 Ethanol/Diethylphthalate (1:3 EtOH:DEP). A vehicle control group was similarly treated using 1:3 EtOH:DEP alone. Four animals were treated in each dose group. The estimated concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was calculated as a percentage dose and µg/cm². A concurrent positive control group was dosed with hexylcinnamaldehyde at concentrations of 5, 10 and 25 % w/v in acetone:olive oil (4:1).

The test material had the capacity to cause skin sensitisation when applied as 25 and 50 % w/v preparations in 1:3 EtOH:DEP. The estimated EC3 value giving rise to a 3 fold increase in lymphocyte proliferation was calculated to be 16.4 % w/v (4100 µg/cm²).

In the positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitisation when applied as a 25 % w/v preparation in acetone: olive oil (4:1 ratio), confirming the validity of the protocol used for this study.

Under the conditions of the study, the test material was considered to be a skin sensitiser.

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

A Local Lymph Node Assay study in the mouse was conducted to investigate the skin sensitisation potential of the test material in accordance with the standardised guidelines OECD 429 and US EPA OPPTS 870.2600 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality set forth by Klimisch et al. (1997).

The test material was applied to the dorsal surface of each ear of female CBA/Ca mice at concentrations of 2.5, 5, 10, 25 and 50 % w/v in 1:3 Ethanol/Diethylphthalate (1:3 EtOH:DEP). A vehicle control group was similarly treated using 1:3 EtOH:DEP alone. Four animals were treated in each dose group. The estimated concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was calculated as a percentage dose and µg/cm². A concurrent positive control group was dosed with hexylcinnamaldehyde at concentrations of 5, 10 and 25 % w/v in acetone:olive oil (4:1).

The test material had the capacity to cause skin sensitisation when applied as 25 and 50 % w/v preparations in 1:3 EtOH:DEP. The estimated EC3 value giving rise to a 3 fold increase in lymphocyte proliferation was calculated to be 16.4 % w/v (4100 µg/cm²).

In the positive control study, hexylcinnamaldehyde was shown to have the capacity to cause skin sensitisation when applied as a 25 % w/v preparation in acetone: olive oil (4:1 ratio), confirming the validity of the protocol used for this study.

Under the conditions of the study, the test material was considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance requires classification with respect to skin sensitisation as Category 1 B (H317; May cause an allergic skin reaction).