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Administrative data

Description of key information

LLNA (OECD 429): sensitising

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
limited data only, no information on positive control / reliability check
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted: 24 April 2002
Deviations:
yes
Remarks:
limited documentation, no information on positive control / reliability check
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted: 22 Jul 2010
Deviations:
yes
Remarks:
limited documentation, no information on positive control / reliability check
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation:
7 - 12 weeks
Vehicle:
dimethylformamide
Concentration:
0.5, 5.0, 10.0, 20.0%
No. of animals per dose:
not specified
Details on study design:
MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
Local Lymph Node Assay
- Criteria used to consider a positive response:
A substance was classified as a skin sensitizer if it induced a threefold or greater increase in local lymph node proliferative activity at one or more test concentrations when compared with concurrent vehicle-treated controls (SI ≥ 3). When the LLNA dose-response curve included concentrations that induced at least one SI greater than 3 and one SI less than 3, EC3 values were calculated by linear interpolation. The EC3 value was used to classify the relative skin sensitisation potency as follows:
EC3 Value (%) ≥ 10 to ≤ 100 -> Weak
EC3 Value (%) ≥ 1 to ≤ 10 -> Moderate
EC3 Value (%) ≥ 0.1 to ≤ 1 -> Strong
EC3 Value (%) < 0.1 -> Extreme

TREATMENT PREPARATION AND ADMINISTRATION:
Animals were exposed topically on the dorsum of both ears to 25 µL of test material or to an equal volume of vehicle alone. Treatment was performed daily for 3 consecutive days. Five days after the initiation of exposure, all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 µCi of tritiated thymidine. Mice were sacrificed 5 hours later, and the draining auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by beta scintillation counting was reported in disintegrations per minute (dpm). A stimulation index (SI) was calculated for each chemical-treated group as the ratio of the dpm of the treated group (or mean dpm when individual animals were assessed) to the dpm or mean dpm of the concurrent vehicle control group.
Positive control substance(s):
not specified
Positive control results:
not specified
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
0.5%
Key result
Parameter:
SI
Value:
3.3
Test group / Remarks:
5.0%
Key result
Parameter:
SI
Value:
5.2
Test group / Remarks:
10%
Key result
Parameter:
SI
Test group / Remarks:
20%
Remarks on result:
not determinable
Key result
Parameter:
EC3
Value:
4.3
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the conditions of the local lymph node assay, the test substance revealed an SI > 3 at 5% and 10% in dimethylformamide. Consequently, the EC3 was calculated to be 4.3%. Therefore, the test substance is considered to be a weak sensitiser.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

The skin sensitising potential of the test substance was investigated in an Local Lymph Node Assay (LLNA) in female CBA mice according to OECD Guideline 429 and in compliance with GLP (Kern, 2010). A volume of 25 µL test substance at concentrations of 0.5, 5, 10, and 20% in dimethylformamide was applied to the dorsum of each ear for 3 consecutive days. Five days after the initiation of exposure, all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 µCi of tritiated thymidine. Mice were sacrificed 5 hours later, and the draining auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The test substance produced a stimulation index of ≥ 3 at concentrations of 5 and 10% and an EC3 value of 4.3% was calculated. Based on the results of this LLNA, the test substance was classified as Skin Sens. 1B.

Since the test substance is classified as Skin Corr. 1, positive results of LLNA (Kern, 2010) might have been biased by skin corrosion. Therefore, additional data based on Bayesian network Integrated Testing Strategy (ITS-3) were considered (Jaworska et al., 2015). The Bayesian network uses information on chemical properties, and experimental data characterising the first 3 key events (DPRA, KeratinoSens and h-CLAT), in the adverse outcome pathway to make a prediction on skin sensitisation potential as measured in the LLNA. In the DPRA prediction, the peptide reactivity represented as percent of free cystein and lysine peptide remaining in the sample was predicted to be 100% and 98.9% for the test substance. Therefore the test substance is considered to show no/low reactivity.

The average concentrations inducing a 1.5-fold or a 3-fold enhanced luciferase activity (KEC1.5 or KEC3.0, respectively) in the transfected HaCaT keratinocyte cell line KeratinoSens™ were predicted to be 62.5 and 1512.0 µM, respectively. The corresponding concentration leading to 50% cytotoxicity after 24 h (IC50) was 1057.8 µM. Therefore, the test substance is considered positive in the KeratinoSensTM prediction (threshold EC1.5 < 1000 µM).

In the h-CLAT prediction, the average test chemical concentrations inducing 150% of vehicle control CD86 cell surface marker expression or 200 % of control cell surface CD54 expression (EC150 or EC200, respectively) in THP-1 cells were predicted to be 223.5 and 245.6 µM. The concentration leading to 25 % cytotoxicity after 24 h (CV75) was determined to be 712.4 µM. Thus, the h-CLAT prediction for activation of dendritic cells is considered positive.

In conclusion, two of three key events were positive for the test substance, confirming the result of skin sensitisation potential for the test substance obtained from the LLNA (Kern, 2010).

Jaworska et al. (2015) Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment: a decision support system for quantitative weight of evidence and adaptive testing strategy. Archives of toxicology 98: 2355-2383

Justification for classification or non-classification

The available data on skin sensitisation meet the criteria for classification according to Regulation (EC) 1272/2008, and the test substance is therefore classified as skin sensitiser Category 1B (H317).