Tetrabutylurea

Endpoint summary

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

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: NMRI / Crl:NMRI

Sex:

female

Details on test animals and environmental conditions:

Species: Mice (non-pregnant, nulliparous, healthy)
Breeder: Charles River Laboratories, Germany
Number of animals: 30 (5 groups of 6 female animals each)
Body weight (on test day 1): 28 - 35 g
Age (on test day 1): 63 days
Adaptation period: At least 5 days
Diet: Commercial diet ssniff® R/M-H V1534. This food was offered ad libitum. Food residue was removed.
Housing: Before application the animals were housed in groups in MAKROLON cages (type III) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 15 cm at a room temperature of 22 +/- 3°C (maximum range) and a relative humidity of 55 +/- 15% (maximum range). After application the animals were housed singly in order to prevent their licking off the test item from the ears of the other animals. The rooms were alternately lit (150 lux at approx. 1.50 m room height) and darkened for periods of 12 hours each.
Drinking water: Tap water was offered ad libitum

Vehicle:

other: acetone/olive oil (3+1, v/v)

Concentration:

0, 25, 50 or 100%

No. of animals per dose:

5 groups of 6 female animals each were examined

Details on study design:

Preparation of the test item: The test item was diluted with acetone/olive oil (3+1, v/v), where appropriate.
Administration: The test item suspension was administered to the dorsum of both animal's ears at an application volume of 25 µL/ear.

Positive control
Route of administration: Open application to the dorsum of both ears
Administration volume: 25 µL/ear
Concentration 25% (v/v)
Positive controls were used to demonstrate appropriate performance of the assay and competence of the laboratory to successfully conduct the assay. An index for the lymph node cell count above 1.4 is considered positive.

Preliminary experiment
A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Three concentrations of 25 and 50% of Tetrabutylurea in acetone/olive oil (3+1, v/v) and the undiluted test item (100%) were examined. Doses were selected according to OECD guideline from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% etc.

Positive control substance(s):

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

Statistics:

yes

Positive control results:

see below

Parameter:

SI

Remarks on result:

other: see table below

Main study results: stimulation indices (SI):

Parameter	Group 1, negative control	Group 2, 25%	Group 3, 50%	Group 4, 100%	Group 5, positive control
Lymph node cell count	1.000	2.714	3.142	3.641	2.008
Lymph node weight	1.000	2.486	3.286	3.371	2.657
Ear weight	1.000	1.136	1.621	1.811	1.095
Difference of ear thickness	1.000	1.102	1.322	1.593	1.157

____ significantly different from control at p ≤ 0.01

 

Interpretation of results:

ambiguous

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item showed strongly irritating properties in this test system as evident from the increased ear weight and ear thickness. Hence, no valid information on possible sensitising properties can be obtained for the test item in the local lymph node assay in mice as the increase in cell count might be caused by the irritating properties.

Executive summary:

The purpose of this study was to determine the sensitising potential of Tetrabutylurea in the local lymph node assay in mice. The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation, as the radioactive method proposed by the OECD guideline led to problems in various EU laboratories: such as (i) practical difficulties/complexity of the test, in particular the radiochemical steps, which sometimes resulted in loss of specimen/activity; this in turn led to variability in the results and to a poor reproducibility and (ii) radiation protection tssues. However, the OECD guideline allows other endpoints for assessment of proliferation in form of lymph node cell counts and lymph node weights if justification and appropriate scientific support exist showing the validity of this method. The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by a European interlaboratory validation exercise. This method has the advantage of (i) more simplistic experimental work, (ii) less variability, (iii) better reproducibility, (iv) faster results, (v) reduced costs. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties. Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method.

Three concentrations of Tetrabutylurea (25% and 50%, w/w) diluted with acetone/olive oil (3+1, v/v) and the undiluted test item were tested in six female NMRI mice per group and compared to a vehicle control group. In addition, a positive control group (25% solution (v/v) of alpha-hexyl cinnamic aldehyde in acetone/olive oil (3+1, v/v)) was employed.

Treatment with Tetrabutylurea at concentrations of 25% and 50 or the undiluted test item revealed statistically significant increased values for lymph node cell count and lymph node weights. However, ear weights and ear thickness were also increased at all 3 concentrations pointing to strongly irritating properties on the mouse ear. The stimulation indices of the lymph node cell count and ear weight exceeded the threshold levels of 1.4 or 1.1. The increase in lymph node weight was concentration-related and significant. The positive control group caused the expected increases in lymph node cell count and lymph node weight. Therefore, the study can be regarded as valid.

No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment. The test item showed strongly irritating properties in this test system as evident from the increased ear weight and ear thickness. Hence, no valid information on possible sensitising properties can be obtained for the test item in the local lymph node assay in mice as the increase in cell count might be caused by the irritating properties.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

1,1,3,3-tetrabutylurea was tested in a Local Lymph Node Assay according to OECD Guideline 429 with GLP compliance. The test item showed strongly irritating properties in this test system as evident from the increased ear weight and ear thickness. Hence, no valid information on possible sensitising properties can be obtained for the test item in the local lymph node assay in mice as the increase in cell count might be caused by the irritating properties.

In an older GPMT, which cannot finally be evaluated (original reference not available), the application of the test substance to the shaved and intact skin caused moderate to no irritation, but no skin sensitisation.

Therefore it can be assumed, that 1,1,3,3-tetrabutylurea has no significant skin sensitisation properties.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

From the available studies it can be assumed, that 1,1,3,3-tetrabutylurea has no significant skin sensitisation properties. Therefore, there is no need for classification and labelling.