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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 19th to May 31st, 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16036C
- Expiration date of the lot/batch: November 2020
- Purity test date:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled Room Temperature (15-25ºC, below 70 RH%)
- Stability under test conditions: stable

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd mice
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (formerly: Harlan Laboratories S.r.l.). San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy.
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Only healthy animals were used for the study. Health status was certified by the veterinarian.
- Age at study initiation: 11-weeks old
- Weight at study initiation: 20.5 – 21.9 g.
- Housing: Group caged, 4 animals/group, mice were provided with glass tunnel-tubes, polypropylene / polycarbonate cages, bedding of certified wood chips.
- Diet: ad libitum ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” (Batch number: 540 5117, Expiry date: 31 July 2016 and Batch number: 278 5652, Expiry date: 30 November 2016) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany).
- Water: ad libitum tap water. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36., Hungary).
- Acclimation period: 28 days.
- Indication of any skin lesions: no.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.1 - 25.3 ºC.
- Humidity (%): 29 - 80 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100% (undiluted), 50% and 25% w/v test item concentration.
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Based on the observation of the solubility test, the maximum available concentration was 100 % (undiluted).
- Irritation: Erythema was observed at the site of the application in the 100 % (undiluted) dose group on Days 2-3 and in the 50 % (w/v) group on Day 3.
- Systemic toxicity: During the Preliminary Irritation / Toxicity Test, no mortality or signs of systemic toxicity were observed. No marked body weight loss (> 5%) was detected on the mean body weight values of the groups; however one animal showed 5.0% body weight loss in the 50 % (w/v) dose group.
- Ear thickness measurements: The ear thickness values and ear punch weights were within the acceptable range. The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups (subjective judgement by analogy with observations of former experiments).
- Erythema scores: Erythema score: 1 was observed at the site of the application in the 100 % (undiluted) dose group on Days 2-3 and in the 50 % (w/v) group on Day 3.

MAIN STUDY
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes). Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses). The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of mice from each test group was pooled and collected in separate
Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
- Clinical observations: During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.
- Body weight: Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay.
- Criteria used to consider a positive response: DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation. Stimulation index (SI = DPN value of a treated group/ DPN value of the negative control group) for each treatment group was calculated. A stimulation index of 3 or greater is an indication of a positive result.

TREATMENT PREPARATION AND ADMINISTRATION: A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C. After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules. After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Stimulation index Positive control (25 % (w/v) HCA in DMSO)= 7.5
DPM Positive control (25 % (w/v) HCA in DMSO)= 2922.6.
Larger than normal lymph nodes were observed in the positive control group.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.8
Test group / Remarks:
100% (undiluted) test item
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
50% w/v test item in acetone:olive oil 4:1 (v:v) mixture
Key result
Parameter:
SI
Value:
0.7
Test group / Remarks:
25% w/v test item in acetone:olive oil 4:1 (v:v) mixture
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: see "Any other information on results" below.

CLINICAL OBSERVATIONS: No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application.

BODY WEIGHTS: In the 100 % (undiluted) dose group tow of four animals had a 5.3 % mean bodyweight loss, however, based on the weight of evidence it is considered that there was no significant systemic toxicity in this 100 % (undiluted) dose group.
In the main test at 100 % (undiluted) concentration two out of four animals had ≥ 5% body weight loss and the mean percentage of the group was also slightly above 5%; however in the preliminary experiment none out of two had marked body weight loss. The other groups had no evidence of treatment related body weight loss or any signs of systemic toxicity. Taking into account the body weight data of the six animals tested at 100 % (undiluted) concentration during the study, this dose is considered to be acceptable, the individual animal weight loss was not considered to be a sign of treatment-related systemic toxicity.

Any other information on results incl. tables

Table 4:DPM, DPN and Stimulation Index Values for all Groups

Test Group Name

Measured DPM / group

DPM

Number
of lymph nodes

DPN

Stimulation Index

Background

31

-

-

-

-

(5 % (w/v) TCA)

33

Negative control

3398

3366.0

8

420.8

1.0

(AOO)

3-Methoxyacetophenone

100 % (undiluted)

2623

2591.0

8

323.9

0.8

3-Methoxyacetophenone

50 % (w/v)

in AOO

3283

3251.0

8

406.4

1.0

3-Methoxyacetophenone

25 % (w/v)

in AOO

2531

2499.0

8

312.4

0.7

Positive control

21777

21745.0

8

2718.1

6.5

(25 % (w/v) HCA
in AOO)

The stimulation index values were 0.8, 1.0 and 0.7 at concentrations of 100 % (undiluted), 50 and 25 % (w/v), respectively.

Table 5:Individual Body Weights for all Animals with Group Means

Animal

Number

Identity

Number

Test Group

Name

Initial Body

Weight (g)

Terminal Body

Weight* (g)

Change#

(%)

1869

1

Negative (vehicle) control

21.7

22.3

2.8

1892

2

AOO

20.9

20.9

0.0

1893

3

 

21.5

22.0

2.3

1900

4

 

21.3

21.0

-1.4

 

 

Mean

21.4

21.6

0.9

1870

5

3-Methoxyacetophenone

21.8

19.9

-8.7

1896

6

100 % (undiluted)

21.2

20.6

-2.8

1871

7

 

21.6

20.7

-4.2

1888

8

 

20.5

19.4

-5.4

 

 

Mean

21.3

20.2

-5.3

1882

9

3-Methoxyacetophenone

21.7

21.7

0.0

1872

10

50 % (w/v)

21.9

20.7

-5.5

1908

11

in AOO

21.0

21.1

0.5

1878

12

 

20.5

21.0

2.4

 

 

Mean

21.3

21.1

-0.6

1884

13

3-Methoxyacetophenone

21.7

21.5

-0.9

1876

14

25 % (w/v)

21.6

20.4

-5.6

1911

15

in AOO

21.7

21.7

0.0

1916

16

 

20.5

19.2

-6.3

 

 

Mean

21.4

20.7

-3.2

1877

17

Positive control

21.5

20.5

-4.7

1899

18

25 % (w/v) HCA

21.7

21.7

0.0

1912

19

in AOO

21.6

20.9

-3.2

1889

20

 

20.9

20.2

-3.3

 

 

Mean

21.4

20.8

-2.8

*: Terminal body weights were measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
Executive summary:

The skin sensitisation potential of the test item was studied according to OECD 429, under GLP conditions. Twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each: three groups received the test item at concentrations of 100 % (undiluted), 50 and 25 % (w/v) in AOO; one negative control group received the vehicle (AOO); and one positive control group received 25 % (w/v) HCA (dissolved in AOO).
The test item solutions were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (
3HTdR) and the values obtained were used to calculate stimulation indices (SI).
No mortality or significant systemic clinical signs were observed during the study. There were no indications of any irritancy at the site of application.
The stimulation index values were 0.8, 1.0 and 0.7 at concentrations of 100 % (undiluted),50 and 25 % (w/v), respectively.

In conclusion, under the conditions of the present assay, the test item, tested in a suitable vehicle, was shown to have no sensitisation potential (non- sensitizer) in the Local Lymph Node Assay. Based on the results, the test item does not need classification according to GHS or CLP criteria.