Cyclohexane, oxidized, aq. ext.

Skin sensitisation

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: Guideline study with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Remarks:

BASF AG, Experimental Toxicology and Ecology

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexane, oxidized, aquous extract

IUPAC Name:

Cyclohexane, oxidized, aquous extract

Constituent 2

Reference substance name:

Cyclohexane, oxidized, aq. ext.

EC Number:

272-810-4

EC Name:

Cyclohexane, oxidized, aq. ext.

Cas Number:

68915-38-8

Molecular formula:

not available

IUPAC Name:

Cyclohexane, oxidized, aq. ext.

Details on test material:

- Name of test material (as cited in study report): Dicarbonsaeure Loesung peroxidfrei EP306/Acid Water
- Analytical purity: Aqueous formulation, content of active ingredient: Residue on drying: 41.8 g/100 g
- Stability under test conditions: The stability of the test item will be verified by reanalysis on completion of exposure/administration or in some other way.

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, Sulzfeld
- Age at study initiation: 8 – 12 weeks
- Weight at study initiation: 16.4 g – 23.2 g
- Housing: single
- Diet (e.g. ad libitum): Kliba-Labordiet
- Water (e.g. ad libitum): Tap water
- Acclimation period: 14 days


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

Study design: in vivo (LLNA)

Vehicle:

other: 1% Pluronic L 92 Surfactant

Concentration:

Undiluted test item with addition of 1% Pluronic (99+1)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- pretest with the undiluted test item with addition of 1% Pluronic L92 were considered, which showed slightly increased lymph node weights and ear weights as indication of ear irritation. Additionally, the pretest showed the feasibility of applying the undiluted test item without producing any toxic effects compromising the outcome of a full study.


TREATMENT PREPARATION AND ADMINISTRATION:
- Test-item preparation and homogenization until end of each application period: The mixture of the undiluted test item + 1% Pluronic (99+1) was produced on a weight per weight basis shortly before the application by stirring with a magnetic stirrer. The homogeneity during application was provided by stirring with a magnetic stirrer.
- Vehicle: 1% Pluronic L 92 Surfactant in highly de-ionized water (control group) and 1% Pluronic L 92 Surfactant (test group)
- Reason for the vehicle: The test item itself is an aqueous preparation. By means of the surface-active agent Pluronic L 92 a run off of the test-item was prevented.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- The study used 1 test group and 1 control group. Each test animal was applied with 25 µL per ear of the test-item 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.
- 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.
- 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.
- Calculations: The stimulation indices of cell count, 3H-thymidine incorporation, lymph node weight and ear weight were calculated as the ratio of the test group values for these parameters divided by those of the vehicle control group.
- Criteria used to consider a positive response: 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.

Positive control substance(s):

other: see free text

Results and discussion

In vivo (LLNA)

Results

Parameter:

SI

Remarks on result:

other: see free text

Any other information on results incl. tables

The stimulation indices (fold of change as compared to the vehicle control) for cell count, 3H-thymidine incorporation, lymph node weight and ear weight are summarized for each test group in the table below.

Treatment	Cell Count SI	3H-thymidine incorporation SI	Lymph Node weight SI	Ear weight SI
vehicle 1% aqueous Pluronic	1.00	1.00	1.00	1.00
test item + Pluronic (99+1) *	1.33	2.47	1.24	1.19

* Calculation on basis of 4 animals, as one animal died during 3H-thymidine injection.

 

- No signs of systemic toxicity were noticed.

- The undiluted test item with addition of 1% Pluronic 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).

- The undiluted test item with addition of 1% Pluronic caused some increase in ear weights as indication of ear skin irritation. Scaling on the ears was observed in all animals on the day of lymph node removal

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

It is concluded that the test substance does not show a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.