2-Propenoic acid, heptadecyl ester, branched

Endpoint summary

Description of key information

LLNA: 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

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

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: 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: 10, 25 and 50%

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 5, 10 and 25% 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 20.5 μCi of 3H-methyl thymidine (equivalent to 82 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Animal no. 6 of the dose group treated with a test item concentration of 10% was accidentally forgotten to be injected with thymidine. The DPM-BG data of this animal was therefore excluded from subsequent calculations and statistical analysis.
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 and the Grubb’s test were 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

Value:

1.18

Test group / Remarks:

10%

Parameter:

SI

Value:

3.77

Test group / Remarks:

25%

Parameter:

SI

Value:

7.14

Test group / Remarks:

50%

Key result

Parameter:

EC3

Remarks:

%

Value:

20.5

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: test substance: 1271.0 (10%); 4047.6 (25%); 7728.0 (50%)

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 and biologically relevant increase in lymph node weights and –cell counts was observed in the animals treated with a test item concentration of 25 and 50% 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 (See Ref. 8). The indices determined for the lymph node cell count of the mid and high dose group (treated with 25% and 50% test item, respectively) exceed this threshold. The index determined for the lymph node cell count of the low dose group (treated with 10% test item) 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 all test item treated dose groups compared to the vehicle control group. The cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice (see Ref. 9) was exceeded in the animals treated with a test item concentration of 25 and 50%. Furthermore, the threshold value of 25% increase in ear weights for excessive local skin irritation mentioned in OECD guideline 429 was slightly exceeded in the high dose group treated with a test item concentration of 50% (mean increase of 29% compared to vehicle control animals). However, the threshold value of 25% increase in ear weights was not exceeded in the mid dose group treated with a test item concentration of 25% (mean increase of 23% compared to vehicle control animals).

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))	1081.6	204.1	1.00
10% C 17 Acrylate	1271.0	273.3	1.18
25% C 17 Acrylate	4074.6	1804.5	3.77^S
50% C 17 Acrylate	7728.0	1403.5	7.14^S

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

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Executive summary:

The test item C 17 Acrylate was found to be a skin sensitiser under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In this study the test item C 17 Acrylate 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 10, 25 and 50% (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 a pre-experiment). In this study Stimulation Indices (S.I.) of 1.18, 3.77 and 7.14 were determined with the test item at concentrations of 10, 25 and 50% (w/w) in acetone/olive oil (4+1, v/v), respectively. A clear dose response was observed. The test item C 17 Acrylate was found to be a skin sensitizer and an EC3 value of 20.5% (w/w) was derived.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the skin sensitization testing, the test item is classified as skin sensitization cat. 1B (H317) according to Regulation (EC) No 1272/2008 (CLP).