Registration Dossier

Administrative data

Description of key information

For the endpoint of skin sensitisation, one murine Local Lymph Node Assay following the OECD 429 TG and GLP compliant (Gerbeix 2010) is available for the substance. The substance was not found to be sensitising in this test.

The absence of any specific reactive functional group (the substance is an hydrocarbon substance) makes respiratory sensitization very unlikely.

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:
03 May 2010 to 19 August 2010
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)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
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: Breeder Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 10 weeks old
- Weight at study initiation: mean body weight  standard deviation of 21.6  1.2 g
- Housing: disposable crystal polystyrene cages
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water (filtered using a 0.22 micron filter)
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From 05 May 2010 to 25 May 2010
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1, 2.5, 5, 10 and 25%
No. of animals per dose:
4 animals per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the test item was soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A solution was obtained at the maximum tested concentration of 50%
- Irritation: Erythema and dryness of the skin of the ears were noted at the concentration of 100% on days 3 and 6, associated with crusts on day 6. Dryness of the skin of the ears and crusts were also noted at the concentration of 50% on day 6. An increase in ear thickness was recorded at the concentrations of 50% and 100%, showing the irritant potential of the test item at these concentrations. The highest concentration retained for the main test was therefore 25%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: study based on design adopted by ICCVAM (Interagency Coordination Committee on the Validation of Alternative Methods, ICCVAM 1999) and ECETOC (Monograph No. 78 Skin sensitization Testing for the Purpose of Hazard Identification and Risk Assessment, September 2000), with the addition of the evaluation of local irritation
- Criteria used to consider a positive response: Stimulation Indice (SI) > or = 3

TREATMENT PREPARATION AND ADMINISTRATION:
Treatment preparation: The test item was prepared at the chosen concentrations in Acetone/olive oil by successive dilutions. The dosage form preparations were homogenized by vortex.
Administration: On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group (SI = 14.09)
Parameter:
SI
Test group / Remarks:
Group 1, vehicle
Remarks on result:
not measured/tested
Parameter:
SI
Value:
1.64
Test group / Remarks:
Group 2: Test item at 1%
Parameter:
SI
Value:
1.3
Test group / Remarks:
Group 3 Test item at 2.5%
Parameter:
SI
Value:
1.79
Test group / Remarks:
Group 4, Test item at 5%
Parameter:
SI
Value:
1.42
Test group / Remarks:
Group 5, Test item at 10%
Parameter:
SI
Value:
2.38
Test group / Remarks:
Group 6, Test item at 25%
Parameter:
SI
Value:
14.09
Test group / Remarks:
Group 7 HCA 25%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
DPM per group: Vehicule: 557.83 Test item at 1%: 912.96 Test item at 2.5%: 727.07 Test item at 5%: 100.80 Test item at 10%: 790.66 Test item at 25%: 1329.78 HCA 25%: 5894.83 DPM per node: Vehicule: 69.73 Test item at 1%: 114.12 Test item at 2.5%: 90.88 Test item at 5%: 125.10 Test item at 10%: 98.83 Test item at 25%: 166.22 HCA 25%: 982.47

No notable lymphoproliferation was noted with the test item at any tested concentration.

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
Under the experimental conditions of this study, the test item BAB did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.
Executive summary:

Methods

A preliminary test was first performed in order to define the concentrations of test item to be used in the main test.

In the main test, twenty eight female CBA/J mice were allocated to seven groups:

. five treated groups of four animals receiving the test item BAB at the concentration of 1, 2.5, 5, 10 or 25% in a mixture acetone/olive oil (4/1; v/v)(vehicle),

. one negative control group of four animals receiving the vehicle,

. one positive control group of four animals receiving the reference item,alpha‑hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25% in the vehicle.

 

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate Stimulation Indices (SI).

 

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

Results

The test item was soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v).A solution was obtained at the maximum tested concentration of 50%.

Consequently, the concentrations selected for the preliminary test were 10, 25, 50 and 100%.

Since the test item was irritant in the preliminary test at the concentrations of 50 and 100%, the highest tested concentration retained for the main test was 25%.

Systemic clinical signs and mortality

Neither treatment-related mortality nor clinical signs were observed during the study.

Local irritation

No cutaneous reactions and no notable increase in ear thickness were observed in the animals of the treated groups.

Proliferation assay

A significant lymphoproliferation was noted in the positive control group given HCA at 25%. The study was therefore considered valid.

No notable lymphoproliferation was noted at any tested concentration.

The results are presented in the following table: 

Treatment

Concentration

(%)

Irritation level

Stimulation Index

(SI)

Test item

1

non-irritant

1.64

Test item

2.5

non-irritant

1.30

Test item

5

non-irritant

1.79

Test item

10

non-irritant

1.42

Test item

25

non-irritant

2.38

HCA

25

-

14.09

Conclusion

Under the experimental conditions of this study, the test item BAB did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the available data on the skin sensitisation, the substance has not to be classified for this hazard class following the classification criteria of the EU Regulation 12/2008 (CLP Regulation). Regarding respiratory sensitisation, there are no data available. Based on the non-sensitising potential for skin sensitisationa and on the absence of structural alerts, it is not expected that the substance could be a respiratory sensitiser.