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Diss Factsheets

Administrative data

Description of key information

The potential of the target substance (87.9% purity) to cause skin sensitisation reactions following topical application to the skin of CBA/JN mice, was assessed using the LLNA: BrdU-ELISA method (OECD 442B). An increase in cell proliferation of draining lymph nodes was observed in the high, medium and low dose groups, indicating that the test item may elicit a sensitisation response.

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:
2015-09-28 to 2016-07-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
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
- Source: Charles River France Laboratories, Domaine des Oncins B.P. 0109, F 69592 L’ARBRESLE CEDEX, France.
- Age at arrival: 8 weeks old
- Weight at arrival: 18 to 19 grams
- Housing: Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm with nesting material
- Diet (e.g. ad libitum): Ad libitum 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/-2
- Humidity (%): 55 +/- 15
- Air changes (per hr): Approximately 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 October 2015 To: 09 November 2015
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Vehicle: 0% w/w (dose group 1),
Test item: 10% w/w (dose group 2),
Test item: 25% w/w (dose group 3),
Test item: 50% w/w (dose group 4),
Positive control: 25% w/w (dose group 5)
No. of animals per dose:
4 females
Details on study design:
RANGE FINDING TEST:
- Irritation: Animals treated for three consecutive days (Days 1, 2, 3) with 25 µL/ear/day of the vehicle or test item formulations at 2.5, 5, 10, 25 and 50%. The treated sites of all animals were examined daily, ear thickness measured by a suitable micrometer on Day 1 (before dosing), on Day 3 (before dosing) and on Day 6. After sacrifice, regularly shaped biopsies obtained from both ears and weighed together.

MAIN STUDY;
- No. of exposures:3
- Test groups: 3 with test item, 1 with positive control
- Control groups: 1 (vehicle)
- Site: Ears, 25 µL/ear/day
- Frequency of applications: once daily
- Duration: 3 days
- Concentrations: 10%, 25%, 50% (Test Item); 25% (Positive control)
- Day 5: intraperitoneal injection of 0.5 mL/animal of a solution of BrdU at a concentration of 10 mg/mL in physiological saline
- Day 6 : Sacrifice, the auricular lymph nodes were excised, pooled on individual basis and individually collected in a solution of 2 % BSA-PBS [2 % bovine serum albumine (BSA) in phosphate buffered saline, PBS]. Cell suspensions were prepared for the evaluation of proliferation. BrdU was measured by ELISA using a commercial kit (Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001, batch no. 10493100). Absorbance (OD) was detected at 450 nm (with reference wavelength: 690 nm).

The BrdU labelling index was calculated for each mouse and a group mean was subsequently calculated. Results for each treatment group were expressed as the mean Stimulation Index (SI). The SI was derived by dividing the mean BrdU labelling index/mouse within each test item group and the positive control groups by the mean labelling indices for the respective vehicle group.

- Criteria used to consider a positive response: The test item is considered to induce sensitisation when the SI for any single treatment dose group is >1.6. It is not required that an increased response is observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Differences between each treated group and the concurrent negative control group (individual BrdU labelling indices) were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test.
Positive control results:
In the group treated with the positive control item, a Stimulation Index of 8.49 was calculated. As it was greater than 2, the study was regarded as valid.
Key result
Parameter:
SI
Value:
4.64
Test group / Remarks:
Dose group 2: 10%
Key result
Parameter:
SI
Value:
4.77
Test group / Remarks:
Dose group 3: 25%
Key result
Parameter:
SI
Value:
3.07
Test group / Remarks:
Dose group 4: 50%
Parameter:
SI
Value:
8.49
Test group / Remarks:
Positive control
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Marked increase in cell proliferation of draining lymph nodes was observed in the high, medium and low dose groups.

DETAILS ON STIMULATION INDEX CALCULATION
BrdU Labelling index/group (OD, Optical Density):
Control: 0.073, Group 2 (10%): 0.338, Group 3 (25%): 0.347, Group 4 (50%): 0.224 and Group 5 (Positive Control): 0.618.
The calculated Stimulation Indices (SI) were 3.07, 4.77 and 4.64, in the high, medium and low dose groups, respectively. The lower SI found at the high dose level is probably due to a lower absorption of test item. In fact, at 50% w/w the formulation formed a paste onto the ears that could easily peeled off. Dunnett’s t test showed a statistically significant difference (p < 0.01) between medium and low dose groups and negative control group.

CLINICAL OBSERVATIONS:
Neither mortality nor clinical signs were recorded in animals treated at all dose levels investigated (10, 25 and 50% w/w).

BODY WEIGHTS:
Changes in body weight observed during the study were within the expected range for this strain and age of animals.

Preliminary test:

-         Five concentrations (50, 25, 10, 5 and 2.5% w/w) of the test item were selected to be used in the preliminary phase.

-         No signs of toxicity (clinical signs or toxicologically relevant body weight losses) were observed at any of the tested concentrations.

-         The evaluation of visible reactions showed no erythema at any of the concentrations investigated (50, 25, 10, 5 and 2.5% w/w).

-         The evaluation of ear thickness indicated that no significant increase was induced by treatment (values of Day 6 compared to Day 1).

-         The evaluation of ear punch weight indicated that no relevant increase was observed in the treated animals, when compared to the animals treated with the vehicle.

 

Based on the results described above, the highest concentration selected for the main assay was 50% w/w.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In an LLNA BrdU-Elisa test (according to OECD 442b) a marked increase in cell proliferation of draining lymph nodes was observed in the high, medium and low dose groups with Stimulation Indices of 3.07, 4.77 and 4.64, respectively. Dunnett’s test showed a statistically significant difference (p < 0.01) between medium and low dose groups and negative control group, indicating that the test item may elicit a sensitisation response.
Executive summary:

In a dermal sensitization study (442B) with the test item (87.9%) four female CBA/JN mice were tested at concentrations of 50, 25 and 10% w/w in acetone/olive oil (4:1 v/v) using the LLNA:BrdU-ELISA method. As positive control hexyl cinnamic aldehyde was used and a stimulation index of 8.49 was calculated indicating the validity of the test system. No mortality nor clinical signs were recorded in any animal. Changes in body weight observed during the study were within the expected range for this strain and age of animals. An increase in cell proliferation of draining lymph nodes was observed in the high, medium and low dose groups with Stimulation Indices of 3.07, 4.77 and 4.64, respectively. Dunnett’s test showed a statistically significant difference (p < 0.01) between medium and low dose groups and negative control group. In this study the test item is a dermal sensitizer (Skin Sens 1, H317). Subcategorisation into 1A/1B is not possible as the EC3 value could not be calculated as the stimulation indices of all concentrations were above 3.

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

The potential of the target substance (87.9% purity) to cause skin sensitisation reactions following topical application to the skin of CBA/JN mice, was assessed using the LLNA: BrdU-ELISA method (OECD 442B). Based on the results from a preliminary test the test item was topically administered at concentrations of 50, 25 and 10% w/w, in acetone:olive oil 4:1 (v/v) in the main assay. No mortality nor clinical signs were recorded in any animal. Changes in body weight observed during the study were within the expected range for this strain and age of animals. An increase in cell proliferation of draining lymph nodes was observed in the high, medium and low dose groups with Stimulation Indices of 3.07, 4.77 and 4.64, respectively. Dunnett’s test showed a statistically significant difference (p < 0.01) between medium and low dose groups and negative control group, indicating that the test item may elicit a sensitisation response.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In a dermal sensitization study according to OECD 442B, female mice were tested positve for the target substance. Based on the results, classification of the target substance is warranted (Skin Sens. 1, H317). Subcategorisation into 1A/1B is not possible as the EC3 value could not be determined as the stimulation indices of all concentrations were above 3.