Cyclohexane, oxidized, non-acidic by-products, distn. residues

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:

2010

Report date:

2010

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 (2010-06-15)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexane, oxidized, non-acidic by-products, distn. residues

EC Number:

271-816-4

EC Name:

Cyclohexane, oxidized, non-acidic by-products, distn. residues

Cas Number:

68609-04-1

IUPAC Name:

(1s,4s)-cyclohexane-1,4-diol (cyclohexyloxy)cyclohexane 2-butylcyclohexan-1-one 8-oxatricyclo[7.4.0.0^{2,7}]trideca-1(9),2(7)-diene cyclohexane cyclohexane-1,3-diol cyclohexanol phenol hydrate

Details on test material:

- Name of test material (as cited in study report): Anone oil
- Analytical purity: The test substance was characterized analytically (for details see analytical report No.: 09L00236).
- Lot/batch No.: 09/0449-1
- 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: 7 – 12 weeks
- Weight at study initiation: 18.5 g – 23.3 g
- Housing: single
- Diet (e.g. ad libitum): Kliba-Labordiet
- Water (e.g. ad libitum): Tap water
- Acclimation period: 7 days


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

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

10%, 30% and 60% w/w preparations of the test substance in propylene glycol or with the vehicle alone

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 10%, 30% and 60% w/w preparations of the test substance in propylene glycol or with the vehicle alone. Each test animal was applied with 25 μL per ear of the respective test-substance preparation to the dorsum of both ears 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.

- The results of a pretest with a 30% test-substance preparation in propylene glycol showed increased ear weights and lymph node weights as possible indication of ear irritation. As no clear signs of ear skin irritation were observed, a 60% preparation was tested as the high concentration. This concentration is an emulsion, already, and well above the effect level of mild to moderate skin sensitizers, leading to a sufficiently stringent conduct of the
study.

Positive control substance(s):

other: see free text

Results and discussion

In vivo (LLNA)

Results

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

When applied as 30% and 60% preparations in propylene glycol, the test substance induced a biologically relevant response in the auricular lymph node cell counts (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) and 3H-thymidine incorporation (increase above the cut off stimulation index of 3). The application of the 10% test-substance concentration caused increases in the auricular lymph node cell counts and 3H-thymidine incorporation into the cells, which failed the cut off stimulation indices of 1.5 and 3, respectively.

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*	1.00	1.00	1.00	1.00
10%	1.39	2.52	1.51	1.13
30%	2.18	5.54	1.77	1.2
60%	2.87	7.89	2.2	1.21

* Calculation on basis of 3 animals, as 2 animals died during 3H-thymidine injection.

• Besides a slight reduction of the mean body weights in test groups 3 and 4, no signs of systemic toxicity were noticed.
• in addition to the inceased stimulation indeces there was a relevant increase in lymph node weights at all concentrations.
• All test-substance concentrations caused some increases in ear weights as indication of ear skin irritation.

Because the lymph node response cannot be fully attributed to the ear skin irritation observed it is concluded that Anone oil shows a skin sensitizing effect in the Murine Local Lymph Node Assay under the test conditions chosen.

The threshold concentration for sensitization induction was >10% <30%.

The estimated concentration that leads to the SI of 1.5 for cell count (EC 1.5) and the estimated concentration that leads to the SI of 3.0 for 3H-thymidine incorporation (EC 3) was calculated

by linear regression from the results of the 10% and 30% concentrations to be 12.8% and 13.2%, respectively.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

Based on the test result the test substance is to be calssified as skin sensitizing according to EU directive 67 (Xi; R43) and GHS.