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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform GLP study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental 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 (lot no. 1114)
- Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control,
microbiological controls at re gular intervals)
- The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding (lot no. 110811)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Based on the results observed in the preliminary test the following test item concentrations were selected for the main study:
6.25%, 12.5% and 25% (w/v)
AOO was used as vehicle and served as negative control.
No. of animals per dose:
5 animals/dose group, 5 animals/control group
3 animals for the preliminary test
Details on study design:
Prescreen Test
Before the initiation of the prescreen test, a solubility test was performed to define the maximum concentration which is technically
applicable to the animals.
The maximum technically applicable concentration of the test item was found to be 25% in AOO (Acetone, Merck, lot no. K42506714,
expiry date: 07/2016; olive oil highly refined, Sigma, lot no. BCBF7133, expiry date: 15/04/2012).
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.
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. Both ears were observed for erythema and scored. 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%.
Two animals were treated by topical application with the test item on three consecutive days at a concentration of 25% diluted with AOO)
to the entire dorsal surface of each ear.
One further animal was treated with 100% AOO 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.
During this period also all clinical signs were recorded.
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 irritation at the application site could be detected in any animal.
All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the duration of the prescreen test.

Preparation of the Animals
The animals were randomly selected.
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
Immediately before the first application the thickness of both ears of all animals was measured. Each mouse was treated by topical
application of 25 µL of the selected solution to the entire dorsal surface of each ear. A second measurement of the ear thickness of all animals
was carried out approximately 48 hours after the first application.
Topical applications were performed once daily over three consecutive days.
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 to a working concentration of 80µCi/mL.
Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. Shortly before sacrificing the thickness of the ears of all animals was measured for a third time. 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.

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 following risk phrases may be assigned in conformity with the criteria given in Commission Regulation (EU) No 286/2011 as well as in GHS - Globally Harmonised System of Classification and Labelling of Chemicals, third revised edition, July 2009:
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: Phenylenediamine
Statistics:
Outlier tests according to Dixon, Grubbs and Nalimov were performed for the values measured for the number of disintegrations per minute (DPM).
If outliers were identified, these values were not included in the calculation of the stimulation indices. As at least four values per group are
required for the evaluation of the results, the outlier test was not repeated to detect further outliers.
Positive control results:
The recent reliability check was performed in February 2012. The raw data of this study are kept in the BSL archives (BSL Project ID 114612 V).
Positive-control substance: P-Phenylenediamine (CAS 106-50-3, Sigma,
purity > 98%; Lot 060M0186V6) 1%
Vehicle: AOO (4:1 (v/v) acetone/olive oil)
Species/strain: healthy CBA/CaOlaHsd mice
Source: Harlan Winkelmann GmbH, 33178 Borchen, Germany
Concentrations: 1% on three consecutive days

The Stimulation Index was 10.9
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
None of the three tested concentrations of the test item reached the stimulation index of 3. The stimulation index at a concentration of 6.25% was 1.2 The stimulation index at a concentration of 12.5% was 1.1 The stimulation index at a concentration of 25.0% was 0.8
Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
As a result of the LLNA test, the substance has no sensitising properties.
Executive summary:

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 were supported by the means of the ear thickness per group, which showed no significant difference compared to the negative control. Thus the substance has no sensitising properties.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed.The body weight of the animals was within the range commonly recorded for animals of this strain and age.


Migrated from Short description of key information:
The sensitization potential of the substance was investigated according to OECD Guideline 429. The test item concentrations chosen for the LLNA test were 6.25, 12.5 and 25 %.

Justification for selection of skin sensitisation endpoint:
Guideline-conform study under GLP without deviations.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance did not show skin sensitizing properties in the Local Lymph Node Assay according to the current OECD guidelines. Allergic skin reactions or case reports of acute contact dermatitis to the substance have not been described in the literature. Therefore, the substance does not have to be classified as a skin sensitizer.