2-Propenamide, 3-(1,3-benzodioxol-5-yl)-N,N-diphenyl-, (2E)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-11-24 to 2010-01-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2002-04-24

Deviations:

yes

Remarks:

only one concentration was used instead of three concentrations

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

, 2008

Deviations:

yes

Remarks:

only one concentration was used instead of three concentrations

Principles of method if other than guideline:

Only one concentration was used instead of three concentrations

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-10-22

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(2E)-3-(2H-1,3-benzodioxol-5-yl)-N,N-diphenylprop-2-enamide

Cas Number:

1309389-73-8

Molecular formula:

C22H17NO3

IUPAC Name:

(2E)-3-(2H-1,3-benzodioxol-5-yl)-N,N-diphenylprop-2-enamide

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age on day 0: 8 - 12 weeks
- Weight on day 0: 17.7 g – 21.3 g
- Housing: single housed in Makrolon cage (type II); nest-building material: wood wool; PLEXX mouse tunnel; bedding: Lignocel PS14
- Diet (ad libitum): Kliba-Labordiät
- Water (ad libitum): tap water
- Acclimation period: 7 days before the first test-substance application

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

Study design: in vivo (LLNA)

Vehicle:

other: 1 % Pluronic® L 92 Surfactant in highly de-ionized water

Concentration:

40 % concentration of the test item (maximum technical applicable concentration)

No. of animals per dose:

5 female mice

Details on study design:

RANGE FINDING TESTS:
- results of a pretest (40 % test-substance preparation in the vehicle): no increase in ear weights or lymph node weights as indication of ear irritation.
- compound solubility: 1 % aqueous Pluronic® was chosen as the vehicle because good homogeneity of the preparation was achieved. By means of the surface-active agent Pluronic® L 92 a run off of the test substance preparations was prevented.

TREATMENT PREPARATION AND ADMINISTRATION:
- test-substance preparation was produced on a weight per weight basis before the application by stirring. The homogeneity of the test-substance preparation during application was also provided by stirring.
- 25 μL were applied to the dorsal part of both ears of the mice on 3 consecutive days (day 1 - 3).
- Day 6: mice were injected intravenously with 20 μCi of 3H-thymidine in 250 μl of sterile saline into a tail vein.
- animals were sacrificed about 5 hours after 3H-thymidine injection.
- circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals and weight of pooled punches was determined for each test group (determination of irritation potential of test item).
- both auricular lymph nodes of each animal were dissected and the weight of the pooled lymph nodes from both sides was determined for each animal.
- pooled lymph nodes of each test group were stored in phosphate buffered saline in an ice water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by passing all lymph nodes per test group through an iron mesh into phosphate-buffered physiological saline.
- determination of cell counts: an aliquot of each suspension was further diluted and lymph node cell count was determined using a counter
- remaining cell suspensions were washed twice with phosphate buffered saline and precipitated with 5 % trichloro-acetic acid. Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a ß-scintillation counter.

- Criteria used to consider a positive response: an increase stimulation indice (SI) of lymph node cell count by a factor of ≥ 1.5 and/or of 3H-thymidine incorporation into the lymph nodes by a factor of ≥ 3 as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test substance.

OBSERVATIONS:
- body weight determination: individual body weights on day 1 prior to the first application and on day 6 prior to the sacrifice of the animals.
- signs and symptoms: obvious signs of systemic toxicity and/or local inflammation at the application sites were noted.
- mortality: twice each workday and once on weekends and public holidays.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Stimulation indices of lymph node cell count, 3H-thymidine incorporationinto the lymph node cells, lymph node weight and ear weight (indicator for irritation potential of test item) were calculated as the ratio of the test group values for these parameters divided by those of the vehicle control group.

Results and discussion

Positive control results:

Stimulation indices (SI) (fold of change as compared to the vehicle control) for lymph node cell count and 3H-thymidine incorporation into lymph nodes is presented below:
Lymph node cell count:
1 % concentration: SI = 1.26
3 % concentration: SI = 1.50
10 % concentration: SI = 1.81
Ratio of test group values to control group values (Stimulation index) greater than 1.5 indicates a positive result

3H-thymidine incorporation:
1 % concentration: SI = 2.18
3 % concentration: SI = 1.48
10 % concentration: SI = 4.82
Ratio of test group values to control group values (Stimulation index) greater than 3.0 indicates a positive result

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Test group / Remarks:

Only 40 % concentration of the test item was tested as the maximum technical applicable concentration

Remarks on result:

not measured/tested

Results 2

Key result

Parameter:

SI

Value:

0.72

Test group / Remarks:

40 % test substance concentration

Remarks on result:

other: A stimulation index of less than 3 was recorded 3H-thymidine incorporation in to the lymph node cells. A stimulation index of less than 1.5 was recorded for the lymph node cell count.

Results 3

Key result

Parameter:

other: disintegrations per minute (DPM)

Value:

934

Test group / Remarks:

40% test substance concentration: 934.0 (dpm/lymph node pair)

Remarks on result:

other: Vehicle: 1292.2 (dpm/lymph node pair)

Any other information on results incl. tables

RESULTS:

- there was no relevant increase in lymph node weights (SI = 1.20)

- test substance preparation did not cause a relevant increase in ear weights as indication of ear irritation (SI = 1.03).

OBSERVATIONS:

- expected body weight gain was generally observed.

- no signs of systemic toxicity were noticed

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an OECD 429 compliant Local Lymph Node Assay, the substance showed no skin sensitising properties and is thus not considered to be a skin sensitiser.

Executive summary:

The skin sensitisation properties of the substance were investigated in female CBA/J mice using the Local Lymph Node Assay (LLNA) according to OECD 429 (2002). A 40 % substance preparation in 1% aqueous Pluronic ® (maximum technically applicable concentration) was used in the study. Furthermore, a vehicle control group was run concurrently. Historical data was used for the positive control substance (85 % α- hexyl cinnamic aldehyde).

A group of five animals was treated with 25 µL/ear of the substance concentration. Another group of five animals was treated with the vehicle.

Treatment was conducted on three consecutive days followed by 3H-thymidine administration on day 5. Five hours following the administration of 3H-thymidine animals were killed. Ear weight (irritation potential of test item), lymph node weight, lymph node cell count and 3H-thymidine incorporation into the lymph node cells were determined for the treatment group as well as for the vehicle control group. The stimulation indices for all parameters were calculated as the ratio of the test group values for these parameters divided by those of the vehicle control group.

Overall, no signs of systemic toxicity were noted and the expected body weight gain was generally observed.

Based on the pooled data the following stimulation indices (SI) were obtained for the 40 % substance concentration:

Lymph node cell count: SI = 1.10

3H-thymidine incorporation lymph nodes: SI = 0.72

Lymph node weights: SI = 1.20

Ear weight: SI = 1.03

With respect to the derived stimulation indices, which were lower than 3 for the 3H-thymidine incorporation and lower than 1.5 for the lymph node cell count, the substance was not considered positive for skin sensitisation properties. The results of the positive control with 85 % α-HCA confirmed the proper conduct of the assay.