Registration Dossier

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:
other: see 'Remark'
Remarks:
GLP guideline study A reduced LLNA test with one concentration was conducted. For this reason, no dose-response information can be obtained. Minor deviations from the guideline were found, but they had no effect on the study results: - incubation period of lymph nodes cells after adding 5% trichloroacetic acid was not stated. - single housing of animals instead of group housing as requested by the guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
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
Qualifier:
according to
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
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2009-10-22
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Physical state: solid/ brown, dark
- Storage condition of test material: room temperature

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)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: A stimulation index of less than 3 was recorded 3H-thymidine incorporation in to the lymph node cells. 40 % concentration: SI = 0.72 A stimulation index of less than 1.5 was recorded for the lymph node cell count. 40 % concentration: SI = 1.10
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle: 1292.2 (dpm/lymph node pair) 40% concentration: 934.0 (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:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The substance is not considered to be a skin sensitiser.
According to the EC-Regulation 1272/2008 and subsequent adaptations, the substance does not require classification as 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 notied 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.

According to the EC-Regulation 1272/2008 and subsequent adaptations, the substance does not require classification as skin sensitiser.