Registration Dossier

Administrative data

Description of key information

skin sensitisation: not 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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
06 July 2012
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0011440677
- Expiration date of the lot/batch: 09.March 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Postbus 6174, 5960 AD Horst / The Netherlands
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 11-12 weeks (in main study)
- Weight at study initiation: 21.2 ± 1.4 g
- Housing: Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet: ad libitum, 2018C Teklad Global 18% protein rodent diet
- Water: ad libitum, tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
methyl ethyl ketone
Remarks:
Purity: 99%
Concentration:
2, 5, 10 %
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS: (5 females used)
- Compound solubility:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used, was 100% of the undiluted test item. Test item solutions at different concentrations were prepared using methyl ethyl ketone (MEK) as vehicle.
- Irritation & Systemic toxicity:
Two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item, on day 3 and before sacrifice the ear thickness was determined. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
- Ear thickness measurements: yes
- Erythema scores: An erythema of the ear skin (score 1-2) was observed in both animals. A further pre-test was performed at 5% in one animal. The animal treated with 5% of the test item did not show any signs of relevant local skin irritation.
MAIN STUDY
The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiments.
TREATMENT PREPARATION AND ADMINISTRATION:
The test item preparations were made freshly and used within two hours before each dosing occasion. The test item in the main study was assayed at 2, 5, and 10%.
Topical application
-each test group of mice treated by topical application to the dorsal surface of each ear (application volume, 25 μL/ear/day, was spread over the entire dorsal surface); once daily for three consecutive days
- control animals treated with an equivalent volume of the relevant vehicle alone
Administration of 3H-methyl-thymidine
-five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.05 μCi of 3H-methyl thymidine (equivalent to 80.2 μCi/mL 3HTdR) injected into each test and control mouse via tail vein
Determination of incorporated 3HTdR
-five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death
-draining lymph nodes were rapidly excised and pooled per animal
-Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation
-lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules
-the precipitates were resuspended in 5 % trichloroacetic acid and transferred to scintillation vials with 10 mL of scintillation liquid
- 3HTdR incorporation measured in a β-scintillation counter
- background 3HTdR levels measured in two 1 mL-aliquots of 5 % trichloroacetic acid
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
All calculations conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count were performed with validated program R Script STABW-mitStat.Rnw). Within the program a statistical analysis conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. Statistical significance was set at the five per cent level (p < 0.05). Additionally, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers.
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Vehicle control
Key result
Parameter:
SI
Value:
1.13
Test group / Remarks:
2% test item
Key result
Parameter:
SI
Value:
1.37
Test group / Remarks:
5% test item
Key result
Parameter:
SI
Value:
1.4
Test group / Remarks:
10% test item
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
A statistically significant increase in lymph node weights and –cell counts was observed in the mid and high dose group in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count determined for all dose groups did not reach or exceed this threshold.
EC3 CALCULATION
The EC3 value could not be calculated, since all SI´s are below the threshold value of 3.
CLINICAL OBSERVATIONS:
No deaths occurred during the study period. No signs of systemic toxicity were observed during the study period. On test days 3 to 6, the animals treated with the high dose of the test item showed a very slight erythema of the ear skin (score 1). On day 6, the animals additionally showed slightly scaly ears.
BODY WEIGHTS
The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Although a statistically significant increase in lymph node weights and –cell counts was observed in the mid and high dose groups in comparison to the vehicle control group, this was not considered to be biologically relevant as the S.I. determined for these concentrations did not reach

or exceed the threshold value of 3. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was not reached or exceeded in any test item treated group (indices of 1.08, 1.40, and 1.50).

Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A GLP-conform Local Lymph Node Assay (LLNA) in mice was performed to assess the skin sensitization potential of the test item according OECD TG 429. The test item was prepared in concentrations of 2, 5, and 10 % diluted in the vehicle methyl ethyl ketone (MEK). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (determined by pre-experiments). As positive control hexyl cinnamic aldehyde dissolved in acetone/olive oil was applied.

The animals neither showed any signs of systemic toxicity nor mortality during the course of the study. Animals treated with the high dose showed very slight erythema of the ear (score 1). A statistically significant increase in ear weights was observed in the high dose group in comparison to the vehicle control group (p<0.05). Furthermore, for BALB/c mice, a cutoff value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. The index determined for the high dose group slightly exceeded this threshold (index of 1.13). However, this was considered to be not biologically relevant, as the observed increase did not reach or exceed the threshold value of 25% for excessive local skin irritation. Stimulation Indices (SI) of 1.13, 1.37 and 1.40 were determined with the test item at concentrations of 2, 5, and 10% (w/w) in methyl ethyl ketone (MEK), respectively. Although a statistically significant increase in lymph node weights and –cell counts was observed in the mid and high dose groups in comparison to the vehicle control group, this was not considered to be biologically relevant as the SI determined for these concentrations did not reach or exceed the threshold value of 3. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was not reached or exceeded in any test item treated group.

In conclusion under the conditions of this study the test item is not considered to be a skin sensitizer (BASF SE 58V0514/15X256; 2016).

 

 

Justification for classification or non-classification

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

The available experimental test data is reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.