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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:
2018-02-26 to 2018-06-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Cerium doped lutetium yttrium orthosilicate
EC Number:
941-731-3
Molecular formula:
Lu2(1-x)Y2xSiO5:Ce, 0
IUPAC Name:
Cerium doped lutetium yttrium orthosilicate
Test material form:
solid
Specific details on test material used for the study:
- Name: LYSO, Ce doped
- Chemical Name: Cerium doped Lutetium Yttrium Orthosilicate
- Nature of Test Substance: crystal (inorganic substance)
- Batch No.: C14-193
- CAS No.: not specified by the sponsor
- Compounds/Purity: 81.25% of Lu2O3, 13.51% of SiO2, 5.2% Y2O3 of and 0.039% of CeO2
- Molecular Weight: 440.7 g/mol
- Physical State: solid (crystal)
- Colour: colourless
- Stability: stable
- Expiry Date: not applicable
- Storage Conditions: at room temperature
- Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Based on the results observed in the prescreen test the following test item concentrations were selected for the main study: 12.5%, 25% and 50% (w/v). The preparations (suspensions) were made immediately prior to each dosing.

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 (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 8-9 weeks
- Housing: The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding.
- Diet (e.g. ad libitum): Ad libitum, Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum): Ad libitum, tap water, sulphur acidified to a pH value of approx. 2.8
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10 x
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
Based on the results observed in the prescreen test the following test item concentrations were selected for the main study: 12.5%, 25% and 50% (w/v)
No. of animals per dose:
5 mice per group, 3 dose groups
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Before the initiation of the prescreen test, a solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals. The maximum technically applicable concentration of the test item in the vehicle was found to be 50% in DMSO.
- Irritation: In order to determine the highest tolerated and not excessively irritant test concentration a prescreen test was performed which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation.
- Systemic Toxicity: The mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination.
- Ear thickness measurements: were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6.
- Erythema scores: Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%.

MAIN STUDY

Control
DMSO was used as vehicle and served as negative control. For animal welfare reasons the negative control was shared. Positive controls are performed periodically.
Other Materials
3H-methyl thymidine (TRK 300, 20 Ci/mmol; PerkinElmer, lot no. 201710E), diluted to a working concentration of 80 µCi/mL.
Phosphate buffered saline (PBS), BSL Munich, lot no. 240118, expiry date: 09/2018.
Trichloroacetic acid (TCA), Sigma-Aldrich, lot no. BCBT4970, expiry date: 19/05/2018.

Preparation of the Test Item
Based on the results observed in the prescreen test the following test item concentrations were selected for the main study:
12.5%, 25% and 50% (w/v)
The preparations (suspensions) were made immediately prior to each dosing.

Preparation of the Animals
The animals were randomly selected using the validated departmental computerised system E WorkBook (latest version, ID Business Solutions Ltd.).
Identification was ensured by cage number and individual marking (tail).

Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

Dose Groups
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.

Test Regime
Topical Application: Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.

Administration of 3H-Methyl Thymidine: Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL.

Preparation of Cell Suspension: Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, weighed, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4 °C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H -Methyl Thymidine: The 3H-methyl thymidine – incorporation was measured in a β-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Evaluation of Results
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3= c+[(3-d)/(b-d)] x (a-c), between two points of the stimulation indices axis, one above (a, b) and one below (c, d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA, if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).
On the basis of the test results, the test substance may be classified into one of the following categories in conformity with the criteria given in Commission Regulation (EU) No 286/201 as well as in GHS - Globally Harmonised System of Classification and Labelling of Chemicals, seventh revised edition, 2017:
Skin sensitiser
Category 1:
A substance is classified as a skin sensitiser
a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or
b) if there are positive results from an appropriate animal test.
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1A:
Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.
EC3 value ≤ 2%
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1B:
Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.
EC3 value > 2%
WARNING, exclamation mark. May cause an allergic skin reaction.
Positive control substance(s):
other:

Results and discussion

Positive control results:
The positive-control substance exceeded the stimulation index of 3 confirming the reliability of the test system.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks:
mean of five animals
Value:
1.3
Variability:
standard deviation = 0.3
Test group / Remarks:
12.5%
Key result
Parameter:
SI
Remarks:
mean of five animals
Value:
1.2
Variability:
standard deviation = 0.3
Test group / Remarks:
25%
Key result
Parameter:
SI
Remarks:
mean of five animals
Value:
1
Variability:
standard deviation = 0.1
Test group / Remarks:
50%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
Please see Table 2 in box "Any other information on results"

EC3 CALCULATION
The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.

CLINICAL OBSERVATIONS:
All animals survived throughout the test period without showing any clinical signs.

BODY WEIGHTS
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

WEIGHT OF LYMPH NODES
The means of the lymph node weights per group showed no relevant difference compared to the negative control. The mean weight of the lymph nodes
for the 50% test group was 3.1 mg
for the 25% test group was 3.1 mg
for the 12.5% test group was 3.5 mg
for the negative control group was 3.3 mg

Any other information on results incl. tables

Results of the pre-screen tests

Irritation: Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any animal.

Cage side observations: Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).

Body weight changes: All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the duration of the prescreen test

Table 2: Results of Main study

POS

Test item

Conc. (%)

SI

MV

Negative control

100

1.0

SD

 

MV

LYSO, Ce doped in DMSO

12.5

1.3

SD

0.3

MV

LYSO, Ce doped in DMSO

25

1.0

SD

0.1

MV

LYSO, Ce doped in DMSO

50

1.0

SD

0.1

MV

Background Szinti and TCA

0

0.0

SD

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, in a mouse local lymph node assay, the test item is not considered to be a skin sensitiser as EC3 values could not be calculated.
Executive summary:

In a dermal sensitisation study conducted according to OECD 429 with LYSO dissolved in DMSO, young adult female CBA/CaOlaHsd mice (5 per dose group) were tested at concentrations of 12.5% (v/v), 25 % (v/v) and 50 % (v/v) in a local lymph node assay (LLNA). Due to animal welfare reasons the negative control was shared and a periodically performed positive control (1% Phenlyenediamine) was used. Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any animal. No mortality was observed in any of the animals. The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3. The results of radioactivity determination are supported by the means of the lymph node weights per group, which showed no relevant difference compared to the negative control. In this study, the test item LYSO, Ce doped is not a dermal sensitiser.