Registration Dossier

Administrative data

Description of key information

LLNA: no indication of skin sensitizing properties (BASF, 2012, OECD 429).

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:
05 October 2011 to 02 November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
24 April 2002
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Batch No.: 110007P040
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CBA/CaOlaHsd: Harlan Laboratories, Horst, Netherlands; CBA/CaCrl: Charles River UK
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 17.4 - 21.6g
- Housing: groups in Makrolon Type II (pretest) or Type III (main study) cages
- Diet: Pelleted standard diet ad lib. (Harlan laboratories, Horst, Netherlands) ad libitum
- Water: tap water ad lib.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C
- Humidity (%): 45-65% (main study)
- Photoperiod (hrs dark / hrs light): 12h/12h
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0%, 1.19%, 2,94%, 6.0% (w/w) (main study - concentration was corrected according to sample analysis)
50%, 100% (1st pre-test)
10%, 25% (2nd pre-test)
2.5%, 5% (3rd pre-test)
No. of animals per dose:
5 (main study)
2 (for each pre-tests)
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: soluble in aceton:olive oil (4:1 v/v)
- Irritation: considered excessive, if erythema ≥3 at any time point or increase in ear thickness ≥ 25% on day 3 or 6
50% / 100% (1st pretest): erythema up to grade 2 and ear swelling of 51% and 81%.
10% / 25% (2nd pretest): erythema up to grade 2 and ear swelling of 33.3% and 102.1%
2.5% / 5% (3rd pretest): erythema grade 1 (day 4-6), only slight ear swelling (app. 8%)
- Lymph node proliferation response: not measured in pretests

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: SI value ≥ 3 and dose response relationship is observed, though local effects or immunological suppression must also be considered.

TREATMENT PREPARATION AND ADMINISTRATION:
25 µL of each concentration was spread evenly onto the dorsal surface of each ear on three consecutive days.
5 days after the first application 250 µL of phosphate-buffered saline (PBS) containing 20.0 µCi of ³HTdR (equivalent to approximately 79.9 µCi/mL ³HTdR) were injected into each test and control mouse via the tail vein. Mice were euthanised 5 hours later.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test was used for identification of possible outliers.
Positive control results:
SI (10%) = 2.18
SI (25%) = 8.08
EC3 = 12.1%
historical control data: SI (25%) = 5.04 - 10.03
Key result
Parameter:
SI
Value:
0.88
Test group / Remarks:
1 % test item
Key result
Parameter:
SI
Value:
0.87
Test group / Remarks:
2.5 % test item
Key result
Parameter:
SI
Value:
1.84
Test group / Remarks:
5% test item
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. No statistically significant increase in lymph node weight was observed in any of the test item treated groups in comparison to the vehicle control group. A statistically significant increase was determined for the lymph node cell counts in the high dose group (p<0.05). The cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was not exceeded in any group.

DETAILS ON STIMULATION INDEX CALCULATION
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

EC3 CALCULATION
The EC3 value could not be calculated, since all S.I.´s are below 3.

CLINICAL OBSERVATIONS:
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.
Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In an LLNA according to OECD 429 and GLP (BASF 2012) the test item dissolved in acetone:olive oil (4+1 (v/v)) was assessed for its skin sensitizing potential using the Local Lymph Node Assay (LLNA) in mice using test item concentrations of 1, 2.5, and 5% (w/w).The highest concentration tested was the highest concentration that could be applied whilst avoiding excessive local skin irritation (as determined by three pre-experiments). The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. A statistically significant increase in ear weights was observed in the high dose group in comparison to the vehicle control group (p >0.05). However, this was considered to be not biologically relevant, as the mean value of the group was still within the range of historical vehicle control data for the ear weight. Furthermore, the cut-off-value for a positive response regarding the ear weight index of 1.1 reported for BALB/c mice was not exceeded in any dose group. In this study Stimulation Indices (S.I.) of 0.88, 0.87, and 1.84 were determined with the test item at concentrations of 1, 2.5, and 5% (w/w) test item in acetone:olive oil (4+1 (v/v)), respectively. A clear dose response was not observed. A statistically significant increase in DPM value and in lymph node cell count was determined in the high dose group in comparison to the vehicle control group (p > 0.05). Still, as the Stimulation Index determined for this group was well below the threshold value of 3 for a positive response, this was not biologically relevant. Also, the cut-off-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was not exceeded in any dose group. The lymph node weights did not show a statistically significant increase in any dose group in comparison to the vehicle control group. An outlier was identified in the low dose group (DPM value determined for animal number 7). However, as the corresponding lymph node weight value confirmed the result of the DPM value and as exclusion of the outlier did not change the overall test result, the value in question was not excluded from calculation.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.