Cyclohexane, oxidized, aq. ext., sodium salt

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: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

QAU BASF SE (2009-09-15)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexane, oxidized, aq. ext., sodium salt

EC Number:

272-812-5

EC Name:

Cyclohexane, oxidized, aq. ext., sodium salt

Cas Number:

68915-39-9

Molecular formula:

Unspecified (UVCB substance)

IUPAC Name:

Cyclohexane, oxidized, aq. ext., sodium salt

Details on test material:

- Name of test material (as cited in study report): Natriumcarboxylat-Loesung
- Analytical purity: Aqueous preparation
- Lot/batch No.: Probe EX 1672
- Stability under test conditions: stable under storage conditions over the study period

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: 19.2 g – 21.1 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 females

Details on study design:

Radioactive Murine Local Lymph Node Assay including measurement of ear thickness.
- Groups of 5 female CBA/J mice each were treated with the undiluted test substance with addition of 1% Pluronic or with 1% aqueous Pluronic alone.
The study used 1 test and 1 control group. Each test animal was applied with 25 μL of the test substance per ear for 3 consecutive days.
- 3 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. 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 Remark

Remarks:

The undiluted test substance 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. Concomitantly, the increase of 3H-thymidine incorporation into the cells was not biologically relevant (no increase above the cut off stimulation index of 3) at this concentration.

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) *	1.13	1.3	1.04	1.11

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

No signs of systemic toxicity were noticed.

There was no increase in lymph node weights, as well.

The test substance caused some increase in ear weights. Minimal test-substance residues were observed on the ears of all animals on study days 1 and 2 and on the day of lymph node removal. One animal showed pull out of hair on the ears on the day of lymph node removal. Due to the test-substance residues the increase in ear weights cannot unequivocally be attributed to ear skin irritation.

In a pretest with the undiluted test substance with addition of 1% Pluronic no increase in lymph node weights was observed. Although, some increase in ear weights as indication of ear irritation was noted. Additionally, the pretest showed the feasibility of applying the undiluted test substance without producing any toxic effects compromising the outcome of a full study.