2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

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:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2002)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2004)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

(2003)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

5,5'-(1H-isoindole-1,3(2H)-diylidene)dibarbituric acid

EC Number:

253-256-2

EC Name:

5,5'-(1H-isoindole-1,3(2H)-diylidene)dibarbituric acid

Cas Number:

36888-99-0

Molecular formula:

C16H9N5O6

IUPAC Name:

5,5'-(1H-isoindole-1,3(2H)-diylidene)dipyrimidine-2,4,6(1H,3H,5H)-trione

Test material form:

solid: particulate/powder

Details on test material:

- Analytical purity: >99%
- Purity test date: (analytical report No.: 08L00151)
- Lot/batch No.: 081012P040
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.

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 at study initiation: 6 – 12 weeks
- Weight at study initiation: 18.5 g – 21.3 g
- Housing: 1 animal per cage (Makrolon cage, type II)
- Diet (e.g. ad libitum): Kliba-Labordiaet (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 7 days

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

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

30% w/w preparations of the test substance in propylene glycol

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The 30% preparation was the maximum technically applicable
concentration.
- Irritation and Lymph node proliferation response: The results of a pretest with a 30% test-substance preparation in propylene glycol were considered for selection of doses/concentrations, which did not show increased ear weights and lymph node weights as indication of ear irritation.

MAIN STUDY
- 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 the test substance 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 substance may induce lymph node responses, the weight of ear punches taken from the area of test-substance application is determined as a parameter for inflammatory ear swelling serving as an indicator for the irritant action of the test substance.

Increase of the stimulation index (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 substance.
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.
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 AND ADMINISTRATION:
25 µl of the test compound was applied to the dorsal part of both ears of each mouse. The application was repeated on day 2 and 3. On day 5 (about 66 to 72 h after the last application) 250 µl sterile saline containing 20 µCi of 3H-thymidine was injected into the tail vein of each experimental mouse. Five hours later, the auricular lymph node of each ear was dissected. The weight of the pooled lymph nodes from both sides was determined for each animal. 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 Casy®ton in a ratio 1:500. The cell count was determined using a Casy®-Counter.
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.

Moreover, a defined area with a diameter of 0.8 cm was punched out of the apical part of each ear and for each test group the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

Positive control substance(s):

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

Statistics:

not reported

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: When applied as 30% preparation in propylene glycol, the test substance caused an S.I. of 2.28, which is below the threshold of 3.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The increase of 3H-thymidine incorporation into the cells (2.28) was not biologically relevant (no increase above the cut off stimulation index of 3).

Any other information on results incl. tables

CELL COUNT, 3H-THYMIDINE INCORPORATION AND LYMPH NODE WEIGHT: TEST GROUP MEAN VALUES AND STIMULATION INDICES

Test Group	Treatment	[Counts/Lymph Node Pair]	Stimulation Index1
1	vehicle propylene glycol	7,617,333	1.00
2	30% in propylene glycol	11,501,333	1.51
Test Group	Treatment	[DPM/Lymph Node Pair]	Stimulation Index1
1	vehicle propylene glycol	607.5	1.00
2	30% in propylene glycol	1,382.8	2.28
Test Group	Treatment	[mg/Lymph Node Pair]	Stimulation Index1
1	vehicle propylene glycol	4.3	1.00
2	30% in propylene glycol	5.3	1.22

EAR WEIGHT: TEST GROUP MEAN VALUES AND STIMULATION INDICES

Test Group	Treatment	[mg/animal]	Stimulation Index1
1	vehicle propylene glycol	32.8	1.00
2	30% in propylene glycol	37.2	1.13

1 test group x / test group 1 (vehicle control), DPM = disintegrations per minute

The test-substance preparation caused minimal increase in ear weights indicating the presence of ear skin irritation. Test substance residues on the ears and orange discolored ears were observed in all animals on study days 1 and 2 and on the day of lymph node

removal.

As the SI for cell count is at the border of biological relevance, the increase of 3H-thymidine incorporation is not biologically relevant and signs of ear skin irritation were observed, the cell count change is not attributed to a skin sensitization reaction.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU