2-Propenoic acid, 2-methyl-, C13-15-branched and linear alkyl esters

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

reduced LLNA

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenoic acid, 2-methyl-, C13-15-branched and linear alkyl esters

EC Number:

292-124-9

EC Name:

2-Propenoic acid, 2-methyl-, C13-15-branched and linear alkyl esters

Cas Number:

90552-04-8

Molecular formula:

C(17-19)H(32-36)O2

IUPAC Name:

2-Propenoic acid, 2-methyl-, C13-15-branched and linear alkyl esters

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 0023067499

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., NL-5961 NM Horst
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.4 - 22.6 g (main test); 16.9 - 22.2 g (pretest)
- Housing: single housing (Polycarbonate cages type MIi with mesh wire tops)
- Diet (e.g. ad libitum): ad libitum (Mouse and rat maintenance diet "GLP", Granovit AG, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: at least 5 days before the first application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

25% (Challenge Treatment: 2.5; 5; 10 and 25% in MEK)

No. of animals per dose:

5

Details on study design:

MAIN STUDY
Randomization: Prior to first application, the animals will be distributed to the individual groups, will receive their animal numbers and will be allocated to the respective cages according to the randomization instructions of WinRando Version 3.2.
Form of application: Epicutaneous application is simulating dermal contact with the compound, which can occur under practical use conditions.
Application volume: 25 μL per ear
Site of application: Dorsal surface of both ears
Frequency of application: 3 consecutive applications (study day 1 - study day 3) to the same application site. Single challenge treatment on study day 21.
3H-thymidine injection: On study day 23 the mice will be injected intravenously (i.v.) with 20 μCi of 3H-thymidine1 in 250 μL of sterile saline into a tail vein of the mice.
Determination of ear weight: lmmediately after sacrifice, a circular piece of tissue (diameter 0.8 cm) will be punched out of the apical part of each ear of all animals. The weight of the pooled punches will be determined
for each animal using an analytical balance. These measurements serve for detecting a potential inflammatory ear swelling.
Removal and weight determination of the lymph nodes: Subsequently, the auricular lymph nodes (right and left) will be dissected. lmmediately after the removal, the total weight of both lymph nodes of each animal will be determined using an analytical balance.
Preparation of cell suspension and determination of cell counts: Both lymph nodes (per animal) will be passed carefully through an iron mesh (mesh size 200 μm) into phosphate-buffered physiological saline. lmmediately afterwards, the cell suspension will be further diluted with isotone and measured by a cell counter straight away.
Measurement of 3H-thymidine incorporation into lymph node cells: The cell suspensions will be washed twice with phosphatebuffered saline (PBS) and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate will be transferred to scintillation fluid and incorporation of 3H-thymidine will be measured by a ß-scintillation counter.
Body weight determination: Individual body weights on study day 1 prior to the first application and prior to sacrifice of the animals on study day 23.
Signs and symptoms: Obvious signs of systemic toxicity will be checked individually during application (study days 1, 2, 3 and 21) and on study day 23, respectively. Local inflammation at the application sites will be checked individually during application (study days 2, 3 and 21) and on study day 23, respectively.
Mortality: A check for moribund and dead animals will be made twice each workday (at the beginning and end of work) and once daily at weekends and on public holidays.

Statistics:

Wilcoxon-Test

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.6

Test group / Remarks:

vehicle MEK / challenge 25% in MEK

Results 2

Parameter:

SI

Value:

2.06

Test group / Remarks:

25% in MEK / challenge 25% in MEK

Results 3

Parameter:

SI

Value:

1.37

Test group / Remarks:

25% in MEK / challenge 10% in MEK

Results 4

Parameter:

SI

Value:

1.45

Test group / Remarks:

25% in MEK / challenge 5% in MEK

Results 5

Parameter:

SI

Value:

1.57

Test group / Remarks:

25% in MEK / challenge 2.5% in MEK

Cellular proliferation data / Observations:

The single application of the 25% test-substance concentration at d21 to the challenge control animals (test group 1) induced statistically significant increases of 3H-thymidine incorporation into the cells and in the auricular lymph node cell counts, which both failed to reach the cut-off (SI 3 for 3H-thymidine incorporation and SI 1.5 for the auricular lymph node cell counts). Additionally, statistically significant increase in lymph node weight was observed in the challenge control group.

The single application of the 25% test-substance concentration at d21 to the animals previously induced with 25% test-substance concentration (test group 2) elicited a statistically significant increase (compared to the vehicle control group) of 3H-thymidine incorporation into the cells of the auricular lymph nodes, which failed to reach the cut-off. A statistically significant (compared to both the vehicle control and challenge control group) just above the cut-off value for auricular
lymph node cell counts was observed. Additonally, a statistically significant (compared to both the vehicle control and challenge control groups) increase of the lymph node weight was noted in this test group.

After the single application of 10%, 5% and 2.5% test-substance concentrations at d21 to the animals to the animals previously induced with 25% test-substance concentration (test groups 3, 4 and 5) no biologically relevant increases in 3H-thymidine incorporation, auricular lymph node cell counts, or lymph node weight was observed. The SI’s were statistically significant (compared to the vehicle control but not the challenge control group) in 3H-thymidine incorporation at 5% and 2.5%, in auricular lymph node cell counts and lymph node weights at 10%, 5% and 2.5%.

DETAILS ON STIMULATION INDEX CALCULATION

EC3 CALCULATION

CLINICAL OBSERVATIONS:
No signs of systemic toxicity were noticed in all animals during general observation.

BODY WEIGHTS
The expected body weight gain was generally observed during the study.
No local findings were observed during the observation period.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).
The test-substance concentrations did not cause an increase (SI ≥ 1.25) in ear weight demonstrating the absence of excessive ear skin irritation. A statistically significant increase was observed in test groups 4 and 5 (compared to both the vehicle control and challenge control groups).

Any other information on results incl. tables

Test Group	Induction Treatment day 1, 2, 3	Challenge Treatment day 21	^3H-thymidine incorporation Stimulation Index^1	Cell Count Stimulation Index^1	Lymph Node Weight Stimulation Index^1	Ear Weight Stimulation Index^1
0	Vehicle MEK	Vehicle MEK	1.00	1.00	1.00	1.00
1	Vehicle MEK	25% in MEK	1.60     #	1.23     #	1.26     ##	1.01
2	25% in MEK	25% in MEK	2.06     #	1.69     ##, **	1.60     ##, *	1.05
3	25% in MEK	10% in MEK	1.37     #	1.30     #	1.40     ##	1.05
4	25% in MEK	5% in MEK	1.45     #	1.34     #	1.43     ##	1.11   #, **
5	25% in MEK	2.5% in MEK	1.57     #	1.28     #	1.36     ##	1.08   #, **

1 test groups 1 to 5 vs. mean of test group 0 (vehicle control)

The statistical evaluations were performed using the WILCOXON-test:

# for p ≤ 0.05, ## for p ≤ 0.01 for comparison vs. mean of test group 0 (vehicle control)

* for p ≤ 0.05, ** for p ≤ 0.01 for comparison vs. mean of test group 1 (challenge control group)

Applicant's summary and conclusion

Conclusions:

Thus, it is concluded that 2-Propenoic acid, 2-methyl-, C13-15-branched and linear alkyl esters did not induce specific lymphocyte proliferation in the Challenge Murine Local Lymph Node Assay (cLLNA) under the test conditions chosen.