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Toxicological information

Skin sensitisation

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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:
other: GLP guideline study.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test item: N-Vinylformamide rein
- Batch identification: Proben-Nummer: 13469
- CAS No.: 13162-05-5
- Purity: 99.5 area-%
- Homogeneity: The test item was homogeneous by visual inspection.
- Storage stability: The stability under storage conditions over the study period was guaranteed by the manufacturer, and the manufacturer holds this responsibility.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 6 - 12 weeks
- Weight at study initiation: 18.2 g – 20.6 g
- Housing: single housing in Makrolon type II cages
- Diet: Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: Tap water ad libitum
- Acclimation period: 7 days


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

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
0% (Vehicle control), 1%, 3% and 10%
No. of animals per dose:
5
Details on study design:
SELECTION OF DOSES/CONCENTRATIONS:
The selection of concentrations took into account available information on the chemical/physical properties, the composition and on acute toxicity and primary irritation/corrosion potential of the test item. In addition results of pretests with 60%, 30% and 10% test-substance preparations in MEK were considered. After the first application of the 60% test-substance preparation in MEK all animals died within 5 hours. The 30% concentration caused mortality in two animals within 24 hours after the first application and the third animal was sacrificed after 24 hours because of poor general state. The 10% testsubstance preparation in MEK showed no increase in ear weights, but slightly increased lymph node weights as indication of ear irritation.
Because of the toxicity of the test substance, the high concentration was limited to 10%.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were
allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 – 64“.
- Criteria used to consider a positive response: In order to reveal a possible induction of sensitization, the response in the draining lymph node after epicutaneous application of several concentrations of the test item to the skin of the ear backs is determined. The parameters used to characterize the response are lymph node cell count, ³H-thymidine incorporation into the lymph node cells and to a certain extent lymph node weight. Because not only sensitization induction but also irritation of the ear skin by the test item may induce lymph node responses, the weight of ear punches taken from the area of test-item application is determined as a parameter for inflammatory ear swelling serving as an indicator for the irritant action of the test item. The increase SI of cell count by a factor of ≥ 1.5 and/or of ³H-thymidine incorporation by a factor of ≥ 3 as compared to the concurrent vehicle control group is generally considered as indicating a sensitizing potential of a test item. If applicable, the EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or using the two nearest points below or above the SI. In addition the evaluation uses the following considerations: If biologically relevant increases in ear weights are running in parallel to the increase in cell count, ³H-thymidine incorporation and/or lymph node weight, it cannot be ruled out, that the lymph node response was caused by irritation and not by skin sensitization. Depending on the magnitude of lymph node response the evaluation of the sensitizing potential may be modified or additional studies might be necessary by expert judgement. If a test substance does not elicit a biological relevant increase in cell count, ³H-thymidine incorporation but shows a clear concentration related increase in response, further investigation of the sensitization potential at higher concentrations should be considered.


TREATMENT PREPARATION:
- Test-item preparation: The test-item preparations were produced on a weight per weight basis shortly before the application. After stirring with a
magnetic stirrer the test item was soluble in the vehicle.
- Vehicle: MEK (methyl ethyl ketone)
- Reason for the vehicle: MEK was used as the vehicle because good solubility of the preparation was achieved.
- Form of application: Solution.


EXPERIMENTAL PROCEDURE:
- Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: No detailed clinical examination of the individual animals was performed but any obvious signs of systemic toxicity and/or local inflammation at the application sites were noted in the raw data.
- Form of application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
- Application volume: 25 μL per ear
- Site of application: Dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- Mortality: Twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays. The animals of control group 1 and test groups 2-4 were treated with vehicle or test-item preparation.
- ³H-thymidine injection: On study day five (about 66 to 72 hours after the last application of test item to the ears) the mice were injected intravenously with 20 μCi of ³H-thymidine in 250 μl of sterile saline into a tail vein.
- Terminal procedures: The animals were sacrificed on study day 5 about 5 hours after ³H-thymidine injection by cervical dislocation.
- Determination of ear weight: Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each test group. These measurements serve for detecting a potential inflammatory ear swelling.
- Removal and weight determination of the lymph nodes: Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
- Preparation of cell suspension and determination of cell count: After weight determination, the pooled lymph nodes of each test group were stored in phosphate buffered saline in an icewater bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing all lymph nodes per test group through an iron mesh (mesh size 200 μm) into 40 mL of phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casyton in a ratio 1:500. The cell count was determined using a Casy-Counter.
- Measurement of ³H-thymidine incorporation of the lymph node cells: The remaining cell suspensions were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of ³H-thymidine into the cells was measured in a ß-scintillation counter.
Positive control substance(s):
other: A concurrent positive control (reliability check) with a known sensitizer was not included into this study. Studies using the positive control substance Alpha-Hexylcinnamaldehyde are performed twice a year in the laboratory.

Results and discussion

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
When applied as 1%, 3% and 10% preparations in MEK, the test item did not induce a biologically relevant response (no increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. There was no relevant increase in lymph node weights, as well. Concomitantly, the increase of ³H-thymidine incorporation into the cells was not biologically relevant (no increase above the cut off stimulation index of 3) at all concentrations.

Any other information on results incl. tables

- Ear weights: All test-item preparations did not cause any increase in ear weights.

- Body weights: The expected body weight gain was generally observed in the course of the study.

- Other findings: No abnormalities were observed during general observation. No signs of systemic toxicity were noticed.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
When applied as 1%, 3% and 10% preparations in MEK, the test item did not induce a biologically relevant response (no increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. There was no relevant increase in lymph node weights, as well. Concomitantly, the increase of ³H-thymidine incorporation into the cells was not biologically relevant (no increase above the cut off stimulation index of 3) at all concentrations. All test-item preparations did not cause any increase in ear weights. Thus it is concluded that N-Vinylformamide rein does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.