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Toxicological information

Skin sensitisation

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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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 22, 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE; Experimental Toxicology and Ecology; Ludwigshafen; Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under nitrogen

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo, The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 19-23.8 g
- Housing: 1 animal/cage
- Diet: ad libitum; Kliba mouse/rat maintenance diet “GLP" by Provimi Kliba SA,Switzerland.
- Water: ad libitum
- Acclimation period: min. 5 days


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
Concentration:
10, 50% (Pretest)
5%, 10%, 50% (Main test)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Yes
- Irritation: Yes
- Systemic toxicity: Yes
- Ear thickness measurements: Yes
- Lymph node weight: Yes

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Prior to first application, the animals were distributed to the individual groups, received their animal numbers and were allocated to the respective cages according to the randomization instructions of „Nijenhuis, A. and Wilf, H.S.: Combinatorial Algorithms, Academic Press, New York, San Francisco, London, 1978, pp. 62 – 64“.

TREATMENT PREPARATION AND ADMINISTRATION:
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: Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal
Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
Form of application: Epicutaneous
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
On study day five (about 66 to 72 hours after the last application of test substance to the ears) the mice were injected into a tail vein with 20 µCi of 3H-thymidine in 250 µL of sterile saline.

- Parameters assessed:
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 animal.

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 animal were stored in phosphate buffered saline (PBS) and a single cell suspension was prepared by carefully passing the lymph nodes through an iron mesh (mesh size 200 µm) into phosphate-buffered physiological saline. 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 with PBS and precipitated with 5% trichloro-acetic acid (TCA). Each precipitate was measured in a β-scintillation counter.

- Criteria used to consider a positive response:
A test item is regarded as a sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of 3H thymidine at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index (SI ≥ 3.0). However, the biological relevance of the obtained stimulation indices are considered in conjunction with the other assessed end points (i.e. cell counts, lymph node weights, ear weights). Hereby, the thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of 1.5 and 1.25, respectively.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
3H-thymidine incorporation, cell count, lymph node weight and ear weight: Wilcoxon - Test

Results and discussion

In vivo (LLNA)

Results
Key result
Parameter:
EC3
Value:
7.6
Test group / Remarks:
3H-thymidine incorporation
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
SI (3H-thymidin-incorporation)
5%: 2.08
10%: 3.85**
50%: 9.85**

SI (cell counts)
5%: 1.16
10%: 1.85**
50%: 2.96**

SI (lymph node weight)
5%: 1.15*
10%: 1.26**
50%: 1.91**

*p<=0.05; **p<=0.01

EAR WEIGHTS
SI:
5%: 0.98
10%: 1.00
50%: 1.04

CLINICAL OBSERVATIONS:
No test substance related signs of systemic toxicity were noticed in all animals during general observation.
Scaling was noted on the ear skin of the animals treated with the 50% test-substance concentration on study day 5.

BODY WEIGHTS:
No relevant influence on the body weights was observed in the course of the study.

Any other information on results incl. tables

 Pretest / Irritation Screening -Results

Signs of systemic toxicity or severe local irritation were not observed in the pretest. The 50% dose-group animal showed very slight erythema on study day 2, only.

However, increased lymph node weights (compared to current vehicle values) were observed in both concentrations.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Executive summary:

The skin sensitizing potential of Tetrahydrolinalool was assessed using the radioactive Murine Local Lymph Node Assay. Groups of 5 female CBA/CaOlaHsd mice each were treated with 5%, 10% and 50% w/w preparations of the test substance in methyl ethyl ketone (MEK) or with the vehicle alone. Each test animal was treated with 25 µL per ear of the appropriate test-substance preparation, applied to the dorsal surfaces of both ears for three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone. Three days after the last application the mice were injected with 3H-thymidine. About 5 hours after the3H-thymidine injection, the auricular lymph nodes were removed and lymph node response was evaluated by measuring3H-thymidine incorporation. Cell counts and weights of pooled lymph nodes from each animal were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation.

When applied as 50% or 10% solution in MEK, the test substance induced a biologically relevant (increase above the cut off Stimulation Index of 3), statistically significant and concentration dependent increase of3H-thymidine incorporation into the cells from the auricular lymph nodes. Moreover these concentrations induced a biologically relevant and statistically significant response (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5) in the auricular lymph node cell counts. Concomitantly, statistically significant increases in lymph node weights were noted at these concentrations and at the 5% concentration. The test-substance concentrations did not cause relevant increases in ear weights, demonstrating the absence of ear skin irritation. Scaling was observed in all animals applied with the 50% concentration on study day 5. No test substance related signs of systemic toxicity was noticed.

Thus, it is concluded that Tetrahydrolinalool exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay underthe test conditions chosen.

The threshold concentration for sensitization induction was >5% <10%. The EC3 for3H-thymidine incorporation and the EC1.5 for lymph node cell counts was found to be 7.6% and 7.4%, respectively.