3,4-dimethoxyphenethylamine

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dimethoxyphenethylamine

EC Number:

204-376-9

EC Name:

3,4-dimethoxyphenethylamine

Cas Number:

120-20-7

Molecular formula:

C10H15NO2

IUPAC Name:

2-(3,4-dimethoxyphenyl)ethanamine

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 50954345T0
- Expiration date of the lot/batch:
- Purity: 99.4 area-% (GC, Rtx-5 Amine capillary), 99.5 area-% (GC, DB-35 MS capillary)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: guaranteed by the sponsor

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V.,Inc., 5960 AD Horst, The Netherlands
- Age at study initiation: ca. 8 weeks (pretest / main test)
- Weight at study initiation: 17.8 g - 19.2 g (pretest) /16.9 - 22.5 g (main test)
- Housing: singel housing (polycarbonate cages type MII)
- Diet (e.g. ad libitum): Kliba mouse/rat maintenance diet “GLP” supplied by Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, (ad libitum)
- Water (e.g. ad libitum): drinking water (ad libitum)
- Acclimation period: at least 5 days before the first application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

10, 25 and 50 %

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The test-substance preparations at different concentrations were solutions in MEK.
- Irritation:
In order to determine the highest test substance concentration that does not induce local signs of skin irritation and/or systemic toxicity a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed. Two mice were treated with test substance concentrations of 1%, 10% and 50% each on three consecutive days.
In the pretest, clinical signs were recorded after each application as well as on day 5. Signs of local irritation were recorded on day 1, 2 and 5. Prior to the first application of the test item (day 0), on day 2 and before sacrifice (day 5) the ear thickness was determined by using a micrometer. Furthermore, the ears were punched after sacrifice at the apical area by using a biopsy punch (Ø 0.8 cm) and were immediately pooled per animal and weighed by using an analytical balance. Additionally, the weight of the pooled lymph nodes from both sides was determined for each animal.
No signs of systemic toxicity were observed in the pretest. At the tested concentrations, the animals did not show relevant signs of local irritation as confirmed by determination of the ear weights (compared to current vehicle values). The ear thickness measurements were only increased at the 50% concentration; however, this may be due to moderate or severe compound residues which were noted on the ear skin during the whole observation period.
Therefore, the following dose levels were selected for the main study: 10%, 25% and 50%.

TREATMENT PREPARATION AND ADMINISTRATION:
The test-substance preparation was produced on a weight per weight basis shortly before application by stirring with a magnetic stirrer. After stirring with a magnetic stirrer, the test substance was soluble in the vehicle. MEK was used as vehicle because good solubility of the preparation was achieved.
Analytical investigation of the test substance preparations was done in compliance with the principles of GLP by the test facitliy Pharmacelsus GmbH, 66123 Saarbrücken, Germany (2017BSF015).
nominal analytical
10%w/w: 9.21%w/w
25%w/w: 23.38%w/w
50%w/w: 54.89%w/w

Positive control substance(s):

other: alpha-Hexylcinnamaldehyde, techn. 85%

Statistics:

Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of 3H thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by that of the vehicle control group.
3H thymidine incorporation, cell count, lymph node weight and ear weight: WILCOXON-Test

Results and discussion

Positive control results:

Historical Positive Control Data for alpha-Hexylcinnamaldehylde (techn. 85%) in MEK are performed twice per year using concentrations of 1%, 5%, 15%. The EC3 (estimated concentration that leads to the SI of 3.0) for 3H thymidine incorporation was calculated by semi-logarithmical regression from the results of all concentrations to be 1.4% and 4.5%, respectively. The EC1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated to be 1.0% and 2.4%, respectively.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Remarks:

3H thymidine incorporation

Value:

5.8

Results 2

Parameter:

other: EC1.5

Remarks:

cell count

Value:

4.6

Results 3

Parameter:

SI

Remarks:

³H-thymidine incorporation

Value:

7.78

Test group / Remarks:

10% in MEK

Results 4

Parameter:

SI

Remarks:

³H-thymidine incorporation

Value:

13.54

Test group / Remarks:

25% in MEK

Results 5

Parameter:

SI

Remarks:

³H-thymidine incorporation

Value:

20.76

Test group / Remarks:

50% in MEK

Cellular proliferation data / Observations:

EC3 / EC1.5 CALCULATION
The threshold concentration for sensitization induction was <10%. The EC3 (estimated concentration that leads to the SI of 3.0) for 3H thymidine incorporation and the EC1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by semi-logarithmical regression from the results of all concentrations to be 5.8% and 4.6%, respectively.

Any other information on results incl. tables

Test Group	Treatment	³H-thymidine incorporation Stimulation Index^1	Cell count Stimulation Index^1	Lymph Node Weight Stimulation Index^1	Ear Weight Stimulation Index^1
1	vehicle MEK	1.00	1.00	1.00	1.00
2	10% in MEK	7.78   ##	1.91  ##	1.44  ##	0.98
3	25% in MEK	13.54   ##	2.71  ##	2.10  ##	1.00
4	50% in MEK	20.76   ##	2.87  ##	2.22  ##	1.12    #

¹test group x/test group 1 (vehicle control)

The statistical evaluations were performed using the WILCOXON-test (# for p≤0.05, ## for p≤0.01)

Pretest / Irritation Screening:

No signs of systemic toxicity were observed in the pretest. After application of the 1%, 10% and 50% concentrations, the animals did not show relevant signs of local irritation as confirmed by determination of the ear weights (compared to current vehicle values). The ear thickness measurements were only increased at the 50% concentration; however, this may be due to moderate or severe compound residues which were noted on the ear skin during the whole observation period.

Main test:

When applied as 10%, 25% or 50% solution in MEK, the test substance induced a biologically relevant (increase above the cut off Stimulation Index of 3), statistically significant and concentration-dependent increase of³H thymidine incorporation into the cells from the auricular lymph nodes. Concomitantly, all concentrations induced a biologically relevant and statistically significant response (increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. 

In addition, statistically significant increases in lymph node weights were noted at all concentrations. The test substance concentrations did not cause increases (SI > 1.25) in ear weight demonstrating the absence of relevant ear skin irritation. However, a statistically significant increase in ear weights was noted at 50%. The expected body weight gain was generally observed during the study. No signs of systemic toxicity were noticed in all animals during general observation. Very slight erythema and/or slight swelling of the ear skin was observed in all animals at the 25% and 50% concentration on study day 2 and/or 5.Depending on the applied concentration slight to severe compound residues were observed on the ear skin in all test-substance treated animals.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

As the EC3 (3H thymidine incorporation) and the EC1.5 (cell count) were calculated to be above 2%, it is concluded that 3,4-Dimethoxyphenethylamine exhibits a skin sensitizing potential (Skin Sens. Cat. 1B according to CLP) in the Murine Local Lymph Node Assay under the test conditions chosen.