Fatty acids, tall-oil, ethoxylated

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 Oct 2008 to 21 Oct 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

April 24, 2002

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

GLP compliance:

yes (incl. QA statement)

Remarks:

Testing Laboratory: Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, ethoxylated

EC Number:

500-150-1

EC Name:

Fatty acids, tall-oil, ethoxylated

Cas Number:

61791-00-2

Molecular formula:

C(18-50)H(34-98)O(3-8)

IUPAC Name:

Fatty acids, tall-oil, ethoxylated

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test substance: AFRANIL T FLUESSIG
- Batch identification: 08007629K0
- Purity: The test substance was characterized analytically

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage stability: The stability under storage conditions over the study period was guaranteed

OTHER SPECIFICS:
- Homogeneity: The test substance was homogeneous by visual inspection.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age on day 0: 6 – 12 weeks
- Body weight on day 0: 18.9 g – 24.1 g
- Housing: 1 animal per cage; Makrolon cage, type II; bedding, Lignocel FS14; SSNIFF
- Diet: Kliba-Labordiät (Maus / Ratte Haltung “GLP”), Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum
- Water: Tap water ad libitum

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

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Remarks:

MEK was used as the vehicle because good homogeneity of the preparation was achieved.

Concentration:

Control group 1: 25 µL methyl ethyl ketone (MEK)
Test group 2: 25 µL of 3% test substance in MEK
Test group3: 25 µL of 10% test substance in MEK
Test group4: 25 µL of 30% test substance in MEK

No. of animals per dose:

5

Details on study design:

TEST-SUBSTANCE PREPARATION
- Test-substance preparation and homogenization until end of each application period: The test-substance preparation was produced on a weight per weight basis shortly before the application by stirring with a magnetic stirrer. The homogeneity of the test-substance preparation during application was provided by stirring with a magnetic stirrer.

EXPERIMENTAL PROCEDURE
- Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: 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 in the raw data.
- Form of application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
- Application volume: 25 μL per ear
- Site of application: Dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site.
- Mortality: Twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
- ³H-thymidine injection: On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected intravenously with 20 μCi of ³H-thymidine in 250 μL of sterile saline into a tail vein.

TERMINAL PROCEDURES
- The animals were sacrificed on study day 5 about 5 hours after ³H-thymidine injection by cervical dislocation.
- Determination of ear weight: Immediately after the death of each animal a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each test group. These measurements serve for detecting a potential inflammatory ear swelling.
- Removal and weight determination of the lymph nodes: Immediately after removal of the ear punches the left and right auricular lymph nodes were dissected. The weight of the pooled lymph nodes from both sides was determined for each animal.
- Preparation of cell suspension and determination of cell count: After weight determination, the pooled lymph nodes of each test group were stored in phosphate buffered saline in an icewater bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing all lymph nodes per test group through an iron mesh (mesh size 200 μm) into 40 mL of phosphate-buffered physiological saline. For determination of cell counts, an aliquot of each suspension was further diluted with Casy®ton in a ratio 1:500. The cell count was determined using a Casy®- Counter.
- Measurement of ³H-thymidine incorporation of the lymph node cells: The remaining cell suspensions were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a ß-scintillation counter.

EVALUATION OF RESULTS
- 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.

Positive control substance(s):

other: Alpha-Hexylcinnamaldehyde

Results and discussion

Positive control results:

Not applicable

In vivo (LLNA)

Results

Parameter:

SI

Value:

< 1.5

Test group / Remarks:

3%, 10% and 30% preparations

Remarks on result:

other:

Remarks:

the test substance did not induce a biologically relevant response in the auricular lymph node cell counts.

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
- When applied as 3%, 10% and 30% preparations in MEK, 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 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) at this concentration.

EAR WEIGHT
The 30% test-substance preparation caused a minimal increase in ear weights as indication of ear skin irritation.

CLINICAL OBSERVATIONS
No abnormalities were observed during general observation.

BODY WEIGHTS
The expected body weight gain was generally observed in the course of the study.

Any other information on results incl. tables

Stability of the test substance preparation:

The stability of the test substance in the vehicle for the maximum application period was confirmed indirectly by analysis of the homogeneity / correctness of the concentration.

Homogeneity of the test substance preparation:

The homogeneity of the test-substance preparation (3%) used for the second application was confirmed by analysis.

Concentration control analysis of the test substance preparation:

The correctness of the concentration of the test-substance preparations (3%, 10% and 30%) for the second application was confirmed by analysis.

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

Test group	Treatment	Cell Count Stimulation Index	³H-thymidine incorporation Stimulation Index	Lymph Node Weight Stimulation Index	Ear Weight Stimulation Index
1	vehicle MEK	1.00	1.00	1.00	1.00
2	3% in MEK	1.00	1.03	1.03	1.01
3	10% in MEK	1.17	1.48	1.09	1.01
4*	30% in MEK	1.28	1.76	1.03	1.08

* Calculation on basis of 4 animals due to irregularities with 1 animal during lymph node removal.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met