Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

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:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: AnLab, Prague, Czech Republic
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 18-24 grams
- Housing: IVC polycarbonate cages, 5 animals per cage, suspended on stainless steel racks in a room equipped with central air-conditioning.
- Diet (e.g. ad libitum):ssniff: s sniff Spezialdiäten GmbH, Germany - ad libitum
- Water (e.g. ad libitum): tap water for human consumption - ad libitum
- Acclimation period: acclimation to conditions identical to test conditions for 5 days prior to start of treatment.
- Indication of any skin lesions: animals were examined by a veterinarian before study initiation. Animals were healthy, without visible signs of disease.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12h light / 12h dark

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
The doses were selected from the concentration series 100 %, 50 %, 25 %, 10 %, 5 %, 2.5 % etc. according to OECD Guideline No. 429.
The starting concentration (25 %) was determined according to pre-screen test result.
No. of animals per dose:
5 females – negative control (vehicle)
5 females – positive control
15 females – test item
4 females - pre-screen test, plus spare animals
Details on study design:
PRE-SCREEN TESTS:
The pre-screen test was conducted under the same conditions as the main LLNA study, except for the assessment of lymph node proliferation.
All mice (2 mice/group) were observed daily for any clinical signs of toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to the termination (day 6). Both ears of each mouse were assessed for any signs of irritation. Ear thickness was measured by a calliper on Day 1 (pre-dose), Day 3 and Day 6. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥ 25%.

MAIN STUDY
Day 1: Each animal was identified and the body weight was recorded. To the dorsum of each ear, 25 µL of the appropriate dilution of the test item, or the vehicle alone was applied.
Days 2 and 3: The application procedure carried out on day 1 was repeated.
Days 4 and 5: No treatment.
Day 6: The body weight of each animal was recorded. 250 µL of sterile phosphate-buffered saline (PBS) containing 2 µCi (7.4 x 10E4 Bq) of 125I-iododeoxyuridine and 10E-5M fluorodeoxyuridine was injected into all test and control mice via the tail vein. Five hours later, the animals were humanely killed. The draining auricular lymph nodes from each ear were excised and pooled in PBS for each experimental group (pooled treatment group approach).

Cell suspension of lymph node cells from pooled treatment groups was prepared by gentle mechanical disaggregation by glass homogenizer. Lymph node cells were centrifuged at 600xg for 10 min at 4°C. Cell suspensions were precipitated with 5 % trichloroacetic acid (TCA) at 4°C for 18h. Pellets were centrifuged at 2000xg for 5 min at 4°C, re-suspended in 1 ml TCA and transferred to gamma counting tubes for 125I-counting.

Incorporation of 125I-iododeoxyuridine was measured by 125I-counting on Automatic Gamma Counter Wizard2 (Perkin Elmer) as counts per minute (CPM). The incorporation is expressed as disintegrations per minute (DPM)/treatment group. DPM were calculated according to formula:

DPM = CPM / absolute detector efficiency

with absolute detector efficiency for Gamma Counter Wizard2 2470 = 66.73 %)

CLINICAL OBSERVATIONS
Animals were carefully observed once daily for any clinical symptoms, either of local irritation at the application site or of the systemic toxicity. All observations were systematically recorded with individual records being maintained for each animal.

BODY WEIGHT
The animal body weights were determined at the start of the test and at the scheduled day of euthanasia of the animals. Significant differences between initial and terminal body weights were analysed by Student´s T-test; p – values less than 0.05 were considered significant.

ERYTHEMA SCORES TABLE
0 = No erythema
1 = Very slight erythema (barely perceptible)
2 = Well-defined erythema
3 = Moderate to severe erythema
4 = Severe erythema (beet redness) to eschar formation preventing grading of erythema

CALCULATION OF RESULTS
Results are expressed as the Stimulation Index (SI). The SI was obtained by dividing the pooled DPM for each treatment group by the incorporation of the pooled DPM vehicle control group; this yields a mean SI. A substance is regarded as sensitizer in the LLNA test when SI≥3.

EC3 value, which induce stimulation indices, is determined by linear interpolation of points on dose-response curve, immediately above and below of SI value, according to the equation:

EC3=c+[(3-d)/(b-d)]x(a-c)

with:
a – higher concentration,
b – SI of higher concentration,
c – lower concentration
d – SI of lower concentration

If all points are below the stimulation index of three, no EC3 value can be stated.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Valid. SI = 4.51

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.21
Test group / Remarks:
25%
Parameter:
SI
Value:
0.93
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
1.51
Test group / Remarks:
100%

Any other information on results incl. tables

Pre-screen test results

The test item administered at concentration of 100% did not cause any changes in monitored parameters. No marked changes of mean body weight were observed during the test. No erythema was observed in both mice after test item administration. On day 1 (pre dose), day 3 and day 6 the ear thickness was measured. The increase in ear thickness did not meet the criteria which are considered as signs for excessive local skin irritation (erythema score ≥ 3; ear thickness increase ≥ 25 %).

Based on the observations recorded in the preliminary test, concentrations of 25, 50 and 100 % were selected for the main test.

Main study test results

The daily clinical observation of the animals did not show visible clinical signs of toxicity. The animal body weights were measured prior to the first treatment and at the scheduled sacrifice. Administration of the test item in used concentrations did not affected thebody weight of treated animals.

In comparison with the control group, a minor increase of the pooled lymph node weights was observed at all used concentrations. The pooled lymph node weights of treated groups were 0.0454 g for 25 % concentration, 0.0408 g for 50 % concentration and 0.0475 g for 100 % concentration of test item. The lymph node weight of control group and positive control group were 0.0406 g and 0.0676 g, respectively. The DPM values for the three treated groups were 2959.49 (25 %), 2292.15 (50 %) and 3717.67 (100 %), respectively. The SI values for the three treated groups were 1.21 (25 %), 0.93 (50 %) and 1.51 (100 %), respectively. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. was not greater than the threshold value of 3.

As a consequence, the test item is not considered a skin sensitizer under the test.

The results are summarized in the below table:

 

Lymph node

Number of

 

 

 

 

weight (g)

lymph nodes

DPM

SI

EC3 (%)

Control

0.0406

10

2454.10

-

-

Positive Control

0.0676

10

11074.84

4.51

Oligomerisation product obtained by plasma treatment of vegetable oil mono- and polyunsaturated C16-C22 triglycerides

25 %

 

0.0454

10

2959.49

1.21

50 %

 

0.0408

10

2292.15

0.93

100 %

 

0.0475

10

3717.67

1.51

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Executive summary:

The sensitization potential of the test item was evaluated using the Local Lymph Node Assay (LLNA) in accordance with OECD guideline 429. The test item was dissolved in DMSO. The positive control (a-Hexylcinnamic aldehyde) (25 %) was dissolved in the same vehicle.

The pre-screen test was performed using the dose of 100 %. Based on the observations recorded in the pre-screen tests, the concentration of 100 % was selected as top dose for the main test.

Five female mice (CBA/Ca) per group were topically exposed (dorsum of both ears) to the test item at concentrations of 25 %, 50 % and 100 %, to the positive control and to the vehicle only. Lymphocyte proliferation was measured using incorporation of radioactive 125I-iododeoxyuridine and 10 -5M fluorodeoxyuridinein the draining lymph nodes. The radioactive incorporation was expressed as disintegrations per minute (DPM)/pooled treatment group and compared with DPM value from the vehicle control group and expressed as the Stimulation Index (SI).

After application of the test item at three concentrations (25 %, 50 % and 100 % v/v) animals did not show visible clinical symptoms of neither local irritation nor systemic toxicity.

In this study the Stimulation Indices (SI) of1.21, 0.93 and 1.51 were determined with the test item at concentrations of 25 %, 50 %, and 100 % in DMSO, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced a SI greater than the threshold value of 3.

The test item Oligomerisation product obtained by plasma treatment of vegetable oil mono- and polyunsaturated C16-C22 triglycerides is not considered a skin sensitizer under the test conditions of this study.