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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:
Aug - Sept 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The relative humidity in the animal room was between aproximately 45 - 85 % and the temperature was 22 +- °C for few hours. This deviation to the study plan, however, does not affect the validity of the study.
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
ANIMALS
- Test system: Mice, CBA/CaOlaHsd
- Rationale: Recommended test system
- Source: Harlan Laboratories B.V.
- Number of animals for the pre-test: 2 females
- Number of animals forthe main study: 16 females
- Number of animals per group: 4 females (nulliparous and non-pregnant)
- Number of test groups: 3
- Number of control (vehicle) groups: 1
- Age: 8 - 12 weeks (beginning of treatment)
- Identification: Single caging. The animals were distributed into the test groups at random and identified by cage number.
- Acclimatisation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRY
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
- Housing: single
- Cage Type: Makrolon Type II, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, D-73494 Rosenberg)
- Feed: pelleted standard diet, ad libitum (Harlan Laboratories GmbH, D-33178 Borchen)
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)
- Environment: temperature 22 + 3°C, relative humidity 45-85%, artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50, 100 %
No. of animals per dose:
4
Details on study design:
Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 25, 50, and 100% (w/v) in acetone:olive oil (4:1). The application volume, 25 µL, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-Methyl Thymidine
Five days after the first topical application, all mice were administered with 250 µL of 81.2 µCi/mL 3HTdR (corresponds to 20.3 µCi 3HTdR per mouse) by intravenous injection via a tail vein.

Determination of Incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium.
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were
prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the
lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.
The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to plastic scintillation vials with 10 mL of ‘Ultima Gold’ scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:
Experiment performed in June 2009, Positive control substance: α-Hexylcinnamaldehyde:
- SI: 5% 1.79 / 10% 2.09 / 25% 6.84
- EC3 = 12.9% (w/v)

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Value:
34.4
Parameter:
SI
Value:
2.11
Test group / Remarks:
25 %
Parameter:
SI
Value:
4.47
Test group / Remarks:
50 %
Parameter:
SI
Value:
4.8
Test group / Remarks:
100 %
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 1137.1, 2407.8, 2588.9 per lymph node Mean DPM/animal values for the experimental groups treated with 25, 50 and 100% were 9097, 19262 and 20711 respectively. The mean DPM/animal value for the vehicle control group was 4312.

Any other information on results incl. tables

All treated animals survived the scheduled study period and no signs of toxicity were observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 2.11, 4.47, and 4.80 were determined with the test item at concentrations of 25, 50, and 100% in acetone:olive oil (4 :1). The EC3 value calculated was 34.4%.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item was found to be a skin sensitiser under the described conditions.
According to the Regulation (EC) No 1272/2008 on classification, labeling and packaging (CLP) of substances and mixtures, the test item should be classified as skin sensitiser (Category 1B).
According to the ECETOC classification scheme for potency (ECETOC Technical Report No. 87, Contact sensitization: Classification according to potency, April 2003, Brussels), the test item would be regarded as weak sensitiser.
Executive summary:

In the study the test item dissolved in acetone:olive oil (4+1) was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 25, 50, and 100%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 2.11, 4.47, and 4.80 were determined with the test item at concentrations of 25, 50, and 100% in acetone:olive oil (4 :1), respectively.

The test item was found to be a skin sensitiser in this assay and an EC3 value of 34.4% was derived.

According to the Regulation (EC) No 1272/2008 on classification, labeling and packaging (CLP) of substances and mixtures, the test item should be classified as skin sensitiser (Category 1B).

According to the ECETOC classification scheme for potency (ECETOC Technical Report No. 87, Contact sensitization: Classification according to potency, April 2003, Brussels), the test item would be regarded as weak sensitiser.