[No public or meaningful name is available]

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Rheinland Pfalz, Germany

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

Santalol oil: fermentation products of glucose with santalene synthase modified Rhodobacter sphaeroides, distilled, oxidised

Cas Number:

2576531-09-2

IUPAC Name:

Santalol oil: fermentation products of glucose with santalene synthase modified Rhodobacter sphaeroides, distilled, oxidised

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

Mouse / CBA/CaOlaHsd / SPF

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS GmbH NL-5961 NM Horst, The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 17.8 g – 20.5 g (pretest); 16.1 g – 20.4 g (main test)
- Housing: single housed
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

other: Ethanol

Concentration:

2.5%, 10% and 25% (w/w)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
In order to determine the highest test-substance concentration that does not induce local signs of skin irritation and/or systemic toxicity, a pretest (experimental conduct in accordance with GLP, but without a GLP status) was performed. Two mice were treated with test-substance concentrations of 10% and 50% each on three consecutive days. Clinical signs were recorded after each application as well as on day 5 and animals were assessed for potential signs of local irritation on day 1, 2 and 5. Prior to the first application of the test item (day 0), on day 2 and before sacrifice (day 5), the ear thickness was determined using a micrometer. Furthermore, the ears were punched after sacrifice at the apical area using a biopsy punch (Ø 0.8 cm) and were immediately pooled per animal and weighed. Additionally, the weight of the pooled lymph nodes from both sides was determined for each animal.
- Compound solubility:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test-substance concentration used in the pretest was a 50% solution in ethanol.
- Irritation:
At the 10% concentration, the animals did not show relevant signs of local irritation as confirmed by determination of the ear weights (compared to historical vehicle values; SI per group = 0.99) and ear thickness measurements. Application of the 50% concentration caused considerably increased ear weight (SI per group = 1.20) just below the border of excessive ear skin irritation in one animal.
- Systemic toxicity:
No signs of systemic toxicity were observed
The lymph node weights were increased in both concentrations (SI per group: 1.70 and 2.34 in 10% and 50% test group respectively).
Based on the results of the pretest, the following dose levels were selected for the main study: 2.5%, 10% and 25% (w/w).

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
- Test-substance preparation: The test-substance preparation was produced on a weight per weight (w/w) basis shortly before application. After stirring with a magnetic stirrer, the test substance was dissolved in Ethanol.
- Reason for the vehicle: Ethanol was used as the vehicle because good solubility of the preparation was achieved.
- Form of application: Epicutaneous application is simulating dermal contact with the compound, which can occur under practical use conditions.
- Application volume: 25 μL per ear
- Site of application: Dorsal surface of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site

INVESTIGATIONS:
- 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.
- ³H-thymidine injection: On study day five (about 66 to 72 hours after the last application of test substance to the ears), ca. 20 μCi 3H-thymidine* in 250 μL sterile saline were injected into the tail vein of the mice.
- Terminal procedures: The animals were sacrificed on study day 5 about 5 hours after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.
- 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. 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 animal were stored in phosphate-buffered saline (PBS) in an ice water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh (mesh size 200 μm) into 8 mL 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 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 β-scintillation counter.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Mean values and standard deviations of the measured parameters were calculated per test group from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated by dividing the mean values per test group and/or single animal values by the mean of the vehicle treated group.
3H-thymidine incorporation, cell count, lymph node weight and ear weight -> WILCOXON - Test.
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 by using the two nearest points below or above the SI.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

3H-thymidine incorporation

Value:

1.47

Test group / Remarks:

2.5% in Ethanol

Results 2

Parameter:

SI

Remarks:

3H-thymidine incorporation

Value:

3.52

Test group / Remarks:

10% in Ethanol

Results 3

Parameter:

SI

Remarks:

3H-thymidine incorporation

Value:

6.7

Test group / Remarks:

25% in Ethanol

Results 4

Parameter:

EC3

Remarks:

3H-thymidine incorporation

Value:

8.1

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation indices (3H-thymidine incorporation)
2.5% in EtOH: 1.47**
10% in EtOH: 3.52**
25% in EtOH: 6.70**
**p<=0.01

Stimulation indices (Cell count)
2.5% in EtOH: 1.34**
10% in EtOH: 1.95**
25% in EtOH: 2.61**
**p<=0.01

EC3 CALCULATION
The threshold concentration for sensitization induction was >2.5% <10%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation and the EC 1.5 (estimated concentration that leads to the SI of 1.5) for cell count was calculated by linear regression from the results of these concentrations to be 8.1% and 4.5%, respectively.

CLINICAL OBSERVATIONS:
No signs of systemic toxicity were noticed in all animals during general observation.

BODY WEIGHTS
The expected body weight gain was generally observed during the study period.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).
No local findings were observed during the observation period.
Stimulation indices (Ear weight)
2.5% in EtOH: 1.00
10% in EtOH: 1.04
25% in EtOH: 1.24**
**p<=0.01