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Description of key information

Based on the results of a LLNA with a NOEL of 3% the substance should be classified with Skin Sens 1B.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
- modified LLNA (IMDS): Measurement of cell counts instead of radioactive labeling. In addition, measurements of ear swelling and ear weights were done to discriminate the irritating potential from the sensitizing potential of the test substance.
Principles of method if other than guideline:
Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Strain: Hsd Win:NMRI
- Age at study initiation: 7 weeks
- Weight at study initiation: 26 - 33 g
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days
Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
The stability of the test item in the vehicle was analytically verified for up to 4 days.
Concentration:
0, 3, 10, 30 %
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation and the vehicle (A/OO) were applied epicutaneously onto the dorsal part of both ears of the animals. This  treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25 µl/ear. The used concentrations were based on the experiences with the test system and the toxic properties of the test substance.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
Investigations:
- weight of draining lymph nodes (given as weight index compared to vehicle controls)
- cell counts in draining lymph nodes (given as cell count index compared to vehicle controls)
Stimulation indices were calculated by dividing the absolute weight or number of cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling (given in 0.01 mm and as index)
- ear weight (given in mg/8 mm diameter punch and as index)
- body weights
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
When it was statistically reasonable, the values from treated groups were compared with those from the control group by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances are considered to be heterogenous (p<=0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5% . Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a probability level of 99% by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differentes in the means were calculated by Scheffels method, which according to Sachs can be used for both equal and unequal sample sizes.
Positive control results:
Alpha hexyl cinnamic aldehyde, checked in regular intervals, shows a clear sensitising potential in the local lymph node assay (IMDS).
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Weight index of draining lymph nodes: statistically increased in the high dose group Cell count index in draining lymph nodes: 'positive level' of 1.4 just exceeded in the mid dose group and clearly increased in the high dose group (statistically significant) Ear swelling: 'positive level' of 2x10E-2 mm increase (i.e. 10% of control level) exceeded in the high dose group (statistically significant) Ear weight: clearly increased in the high dose group (statistically significant)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: modified LLNA; measurement of cell counts instead of radioactive labeling

Results of LLNA (means for 6 animals per group)

 

Parameters investigated

Vehicle

Low (3% in A/OO)

Medium (10% in A/OO)

High (30% in A/OO)

Stimulation Index: weight of draining lymph nodes

 1.00

 0.87

 1.32

 1.61*

 Stimulation index: cell count in draining lymph nodes   1.00   0.90 1.41   2.03*
 Ear swelling in 0.01 mm on day 4 (index)   18.00 (1.00)   18.25 (1.01) 19.00  (1.06)   27,17* (1.51)
 Ear weight in mg/8 mm diameter punch on day 4 (index)   11.76 (1.00)   11.69 (0.99)   13.21 (1.12)   16.53* (1.41)

* statistically significant increase (p<= 0.05)

Body weights were not affected by treatment.

In the high dose group ear swelling and ear weighs were significantly increased in the high dose group.

Compared to vehicle-treated animals there were clear increases in weight and cell counts of the draining lymph nodes in the high dose group. These increases were of statistical significance. The 'positive level' index of 1.4 for cell counts was strongly exceeded in the high dose group and just exceeded in the mid dose group. A sensitising potential can therefore be asssumed. The differentiation index (DI) was calculated, which is the quotient of the relative lymph node reaction divided by the relative acute skin reaction. For the high concentration a DI of < 1 (i.e. 0.5) was determined, pointing to an overall irritating potential at this concentration. For the mid concentration (10%) a DI of > 1 (i.e. 1.14) was determined, also pointing to a skin sensitising potential at this concentration.

The EC 1.4 value was calculated with 0.86% for the test item. In accordance with the Techical Report No. 78 of ECETOC (Brussels, 2003) this value corresponds to a moderate skin sensitiser.

Besides a non-specific irritant poperty the test item has to be considered as moderate skin sensitiser. The NOEL was determined with 3% for the parameters investigated in this study.
Interpretation of results:
other: moderate skin sensitiser
Executive summary:

The test substance was investigated in the modified Local Lymph Node Assay (LLNA) on female mice, performed according to OECD TG 429, in concentrations of 0% (vehicle control), 3%, 10%, and 30% in acetone/olive oil (4:1). Body weights were not affected by treatment. In the high concentration group all investigated parameters (weight and cell count in draining lymph nodes, ear swelling and ear weight) were significantly increased compared to vehicle controls. In the mid concentration group the 'positive level' index of 1.4 for cell counts was just exceeded.

Besides a non-specific irritant property of the test item, a specific activation of the cells of the immune system via dermal route was determined after application of 30% by the method used. The test substance therefore has to be classified as a moderate skin sensitiser in mice after dermal application. The concentration of 3% turned out to be the NOEL for the parameters investigated in this study.

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

The test substance was investigated in the modified Local Lymph Node Assay (LLNA) on female mice, performed according to OECD TG 429, in concentrations of 0% (vehicle control), 3%, 10%, and 30% in acetone/olive oil (4:1). Body weights were not affected by treatment. In the high concentration group all investigated parameters (weight and cell count in draining lymph nodes, ear swelling and ear weight) were significantly increased compared to vehicle controls. In the mid concentration group the 'positive level' index of 1.4 for cell counts was just exceeded.

Besides a non-specific irritant property of the test substance, a specific activation of the cells of the immune system via dermal route was determined after application of 30% by the method used. The test substance therefore has to be classified as a moderate skin sensitiser in mice after dermal application. The concentration of 3% turned out to be the NOEL for the parameters investigated in this study and thus the substance should be classified with Skin Sens Cat 1B.

No read-across of Bisphenol A data is appropriate for skin sensitization. Although a LLNA with Bisphenol A showed no skin sensitizing effects Bisphenol A is classified as Skin Sens. 1 according to Annex VI of Regulation (EC) No 1272/2008. The EU-RAR update 2008 concluded with regard to this LLNA: "Overall the new information does not confirm the previously reported evidence of a skin sensitization potential of Bisphenol A. While the data do not exclude a skin sensitizing activity of Bisphenol A at high concentrations (> 30%), there is no evidence that this is a concern for workers in current Bisphenol A manufacturing plants (such workers are believed to represent the group most likely to be exposed to Bisphenol A dust)."

Due to the low if any skin sensitization potential of Bisphenol A and the clear LLNA result as Cat 1B skin sensitizer for the UVCB substance itself, the UVCB substance will be classified with Skin Sens 1B.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Besides a non-specific irritant property of the test item, a specific activation of the cells of the immune system was determined in the LLNA after dermal application of a 30% concentration in acetone/olive oil (4:1). The NOEL turned out to be 3%. The test item has thus to be classified as a moderate skin sensitiser category 1B (Skin Sens 1B, H317) after dermal application according to 1272/2008 EC and Commission Regulation 286/2011 EC.

No data are available for respiratory sensitisation.