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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
Study period:
August 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)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Name: RCX 15-116
CAS No.: 15305-07-4
Batch No.: 15047AE2
Purity: > 99%
Chemical Name: Aluminium-N-nitrosophenylhydroxylamine (NPAL)
Aggregate State at RT:solid
Colour: whitish
Storage Conditions: room temperature, protected from light
Stability: Instable after repeated contact to air and / or light, undergoes hydrolysis
Expiry Date: 30 Nov 2016
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Species/strain: healthy CBA/CaOlaHsd mice
Source: Envigo (formed partially from Harlan in September 2015), 5800 AN Venray, The Netherlands
Sex: female (nulliparous and non-pregnant)

Age at the beginning of the study: 8-9 weeks
Number of animals: 5 mice / group
5 mice / prescreen test

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least five days) under laboratory conditions

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
Based on the results observed in the pre-screen test the following test item concentrations were selected for the main study: 6.25%, 12.5% and 25% (w/v). The preparations were made immediately prior to each dosing.
No. of animals per dose:
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.
Details on study design:
PRE-SCREEN TESTS:

- Compound solubility: before the initiation of the pre-screen 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 25% in DMSO.
- Design of pre-test: in order to determine the highest tolerated and not excessively irritant test concentration a pre-screen test was performed which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation. four animals were treated by topical application with the test item on three consecutive days at the following concentrations to the entire dorsal surface of each ear: Animals no. 1 and no. 2 were treated with a test item concentration of 25% (suspended with DMSO). Animal no. 3 and no. 4 were treated with a test item concentration of 12.5% (suspended with DMSO). One further animal was treated with 100% DMSO and served as negative control. Immediately before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of all animals was measured. The mice were observed daily or local irritation at the application site.
- Systemic toxicity: the mice were observed daily for any clinical signs of systemic toxicity. Body weights were recorded pre-test and prior to termination. 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). Neither signs of systemic toxicity nor signs of excessive irritation at any application site could be detected in any animal.
- Ear thickness measurements: Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6. Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%.
- Erythema scores: no signs of excessive irritation at any application site could be detected in any animal.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymp Node Assay
- Criteria used to consider a positive response:

TREATMENT PREPARATION AND ADMINISTRATION:

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, 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 ß-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.
Positive control substance(s):
other: 1% phenylenediamine
Statistics:
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.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
5.3
Test group / Remarks:
6.25%
Key result
Parameter:
SI
Value:
5.7
Test group / Remarks:
12.5%
Key result
Parameter:
SI
Value:
4.5
Test group / Remarks:
25%

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on the results of the LLNA test, the substance should be considered as a skin sensitiser.
Executive summary:

Based on the results of the LLNA test, the substance should be considered as a skin sensitiser. Each of the tested concentrations exceeded the stimulation index of 3. The EC3 value could not be calculated dueto the lack of a dose-response relationship of the data obtained.