Registration Dossier

Administrative data

Description of key information

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:
OECD Guideline 429 (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: Harlan Laboratories B.V., Horst / The Netherlands
- Age at study initiation: 1st pre-test: 8 - 9 weeks (beginning of treatment); 2nd pre-test: 10 -11 weeks (beginning of treatment); Main study: 8 - 9 weeks (beginning of treatment)
- Housing: goup; Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): approx. 45-65%
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Main test: 2, 5 and 10 %
No. of animals per dose:
5
Details on study design:
Methode
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2, 5 and 10% in acetone:olive oil (4+1, v/v). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.2 μCi of 3H-methyl thymidine (equivalent to 80.8 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death. After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A statistical analysis was 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. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as a 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 detection of possible outliers (performed with Microsoft Excel 2007). However, both biological and statistical significance were considered together.
Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in April 2014.
Parameter:
SI
Remarks on result:
other: In this study Stimulation Indices (S.I.) of 1.26, 1.28 and 2.46 were determined with the test item at concentrations of 2, 5 and 10% (w/w) in acetone:olive oil (4+1, v/v), respectively. A clear dose response was not observed.

Lymph Node Weights and Cell Counts

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weights or cell counts was not observed in any of the test item treated groups 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 did not exceed this threshold.

Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A statistically significant increase in ear weights was observed in the animals treated with a test item concentration of 10%. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was exceeded in the group treated with a test item concentration of 10%. However, the threshold value of 25% increase in ear weights for excessive local skin irritation mentioned in OECD guideline 429 was not exceeded in any group.

Test item concentration

Group Calculation

Mean DPM per animal (2 lymph nodes)a)

SD

S.I.

Vehicle Control Group (acetone:olive oil (4+1, v/v))

832.9

316.3

1.00

2% C 17 Methacrylate

 

1049.5

411.7

1.26

10% C 17 Methacrylate

 

1069.1

449.3

1.28

25% C 17 Methacrylate

 

2052.7

864.7

2.46S

a)     Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

S  Mean DPM value for the group was according to the ANOVA (Dunnett-test) significantly higher than the corresponding control value. The p value for the analysis was p<0.05

Executive summary:

The test item C 17 Methacrylate was not a skin sensitiser under the test conditions of this study.

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

In this study the test item C 17 Methacrylate was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice. Test item solution at different concentrations was prepared in the vehicle acetone:olive oil (4+1, v/v). For this purpose a local lymph node assay was performed using test item concentrations of 2, 5 and 10% (w/w). The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by two pre-experiments). In this study Stimulation Indices (S.I.) of 1.26, 1.28 and 2.46 were determined with the test item at concentrations of 2, 5 and 10% (w/w) in acetone:olive oil (4+1, v/v), respectively. A clear dose response was not observed. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3. The test item C 17 Methacrylate was thus not a skin sensitiser under the test conditions of this study.


Migrated from Short description of key information:
LLNA: not sensitising (BASF, 2014)

Justification for selection of skin sensitisation endpoint:
Key study

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available studies data on skin sensitisation properties the test item does not have to be classified and labelled according to Regulation (EC) No 1272/2008 (CLP).