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

Endpoint summary

Description of key information

Two test materials by two different suppliers were tested for skin sensitization in the LLNA (GLP, OECD 429) in the years 2005 and 2008.

One test material was a nanomaterial (no batch-specific analysis available), for the other test material such information coculd not be retrieved.

Neither test material was found to be a skin sensitizer in the LLNA.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2002)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2004)

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)

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

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

Parameter:

SI

Remarks on result:

other: When applied as 30% preparation in propylene glycol, the test substance showed a simulation index for 3H-thymidine incorporation of less than 3.

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).

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.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There are reliable studies available to assess the skin sensitising potential of the test substance.

The skin sensitising potential of the test substance (purity: > 99 %) was assessed using the radioactive Murine Local Lymph Node Assayin a GLP conform study conducted according to OECD guideline 429 (BASF SE, 2008). Based on the trade name of the substance in the study report, the test material was a nanomaterial. However, the actual sample has not been characterized for particles size distribution. Groups of 5 female CBA/J mice were treated with a 30% w/w preparation of the test substance in propylene glycol or with the vehicle alone. The 30% test-substance preparation was the maximum technically applicable concentration. The study used 1 test group and 1 control group. Each test animal was applied with 25 μL per ear of the respective test-substance preparation to the dorsum of both ears for three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone. Three days after the last application the mice were injected intravenously with 20 μCi of ³H-thymidine in 250 μL of sterile saline into a tail vein. About 5 hours after the ³H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. The weights of each animal’s pooled lymph nodes were determined. Thereafter lymph nodes were pooled group wise and further evaluated by measuring their cellular content and ³H-thymidine incorporation into the lymph node cells (indicators of cell proliferation). 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.

The stimulation indices (fold of change as compared to the vehicle control) for cell count, ³H-thymidine incorporation, lymph node weight and ear weight were examined. No signs of systemic toxicity were noticed. When applied as 30% preparation in propylene glycol, the test substance induced increased auricular lymph node cell counts (SI = 1.51) at the border of biological relevance(increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5). In addition, there was some increase in lymph node weights. Contrary to the cell count, the increase of ³H-thymidine incorporation into the cells of 2.28 was not biologically relevant (no increase above the cut off stimulation index of 3) at this concentration.

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.

Thus it is concluded that the test itemdoes not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

 

In another LLNA the test item was assessed for its possible contact allergenic potential in a GLP conform study conducted according to OECD guideline 429 (RCC 2005). For this purpose three groups each of 4 female mice were treated daily with 25 μL of the test item at concentrations of 7.5, 15 and 30 % (w/v) dissolved in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methylthymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a ß-scintillation counter.

All treated animals survived the scheduled study period. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices (S.I.) of 1.4, 1.1, and 1.1 were obtained with the test item at concentrations of 7.5, 15, and 30 % (w/v), respectively. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than 3 (cut off value for a positive sensitizer).

Under the conditions of this study the test substance does not show a skin sensitizing effect in the Murine Local Lymph Node Assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No increase in the stimulation index was observed in the LLNA (OECD 429). Therefore, the substance does not require classification as a skin sensitizer under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.