N,N''-(isobutylidene)diurea

Endpoint summary

Description of key information

The test substance was evaluated in the Local Lymph Node Assay (LLNA) according to OECD Guideline 429 (BASF SE, 2009). IBDU does not show a skin sensitization potential in this assay.

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:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 6 – 12 weeks
- Weight at study initiation: 18.2 g – 21.3 g
- Housing: single housed
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C
- Humidity (%): 30 – 70%
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Vehicle:

propylene glycol

Concentration:

50% in propylene glycol

No. of animals per dose:

5 females

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The 50% preparation was the maximum technically applicable concentration.
- Irritation: very slight irritation was observed in the range finding test at a 50 % concentration but not in the main study at the same concentration
- Lymph node proliferation response: no biologically relevant response was observed


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA according to OECD 429 guideline
- 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 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.
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 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 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 AND ADMINISTRATION:

Positive control substance(s):

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

Statistics:

not applicable

Key result

Parameter:

SI

Test group / Remarks:

50%

Remarks on result:

other: see Remark

Remarks:

When applied as 50% preparation in propylene glycol, the test substance 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 increase in lymph node weights, as well.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: vehicle control (propylene glycol): 418.3 dpm/lymphnode pair 50% in propylene glycol: 657.8 dpm/lymphnode pair

The skin sensitizing potential of Isodur was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of the auricular lymph nodes on repeated application of the test substance to the dorsal skin of the ears. Groups of 5 female CBA/J mice each were treated with a 50% w/w preparation of the test substance in propylene glycol or with the vehicle alone. The 50% 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 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.

No signs of systemic toxicity were noticed. When applied as 50% preparation in propylene glycol, the test substance 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 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).

The test-substance preparation did not cause any increase in ear weights. Thus it is concluded that Isodur does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitizing potential of IBDU was assessed using the radioactive Murine Local Lymph Node Assay according to OECD guideline 429 (BASF SE, 2009). The assay simulates the induction phase for skin sensitization in mice. It determines the response of the auricular lymph nodes on repeated application of the test substance to the dorsal skin of the ears. Groups of 5 female CBA/J mice each were treated with a 50% w/w preparation of the test substance in propylene glycol or with the vehicle alone. The 50% 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 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.

 

When applied as 50% preparation in propylene glycol, the test substance 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 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).

 

The test-substance preparation did not cause any increase in ear weights. Thus it is concluded that IBDU does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.  

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

This information is not available

Justification for classification or non-classification

IBDU was negative in a LLNA according to OECD guideline 429 (BASF SE, 2009). Therefore no classification for a sensitizing potential is required.