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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
calculation (if not (Q)SAR)
Remarks:
Migrated phrase: estimated by calculation
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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
Details on test material:
- Name of test material (as cited in study report): Golpanol ALS wasserfrei
- Analytical purity: 71.3%
- Lot/batch No.: 04749356P0
- Expiration date of the lot/batch: 2010-09-05
- Stability in solvent: The stability of the test substance in the vehicle was verified indirectly by concentration control analysis
- Storage condition of test material: At room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V. Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 20.5 +- 1.3 g
- Housing: Single caging (Makrolon Type II, with wire mesh top)
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst / Netherlands)
- Water (e.g. ad libitum): Tap water, ad libitum, (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2°C
- Humidity (%): 20-65%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12:12h

Study design: in vivo (LLNA)

Vehicle:
other: ethanol:sterile water (3+7)
Concentration:
0 (vehicle control), 5, 10, 25%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which can be technically used was a 25% (w/v) solution in ethanol:sterile water (3+7). Vortexing was necessary to prepare the solution. Before use, it was necessary to shake the vessel containing the test item.
- Irritation: In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 6.
- Lymph node proliferation response: only ear thickness and ear weight was measured

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.


TREATMENT PREPARATION AND ADMINISTRATION:
Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 5, 10, and 25% (w/v) in ethanol:sterile water (3+7). The application volume, 25 µl, was spread over the entire dorsal surface (diameter ca. 8 mm) of each ear 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
3H-methyl thymidine (3HTdR) was purchased from GE Healthcare (specific activity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 µl of 80.9 µCi/m 3HTdR (corresponds to 20.2 µCi 3HTdR per mouse) by intravenous injection via a tail vein.
Determination of lymph node weights
For the determination of lymph node weight, after excision, the lymph nodes were pooled per animal and weighed immediately after removal using an analytical balance.
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 beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
Determination of Ear Weights
After the lymph nodes have been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (diameter 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance.
Interpretation of Raw Data
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (Stimulation Index; S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
The ANOVA (Dunnett-test) was conducted on the ear and lymph node weights to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 5 %: 0.76 10 %: 0.70 25 %: 0.45
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 0 %: 1900 5 %: 1444 10 %: 1323 25 %: 853

Any other information on results incl. tables

The EC3 value could not be calculated, since all S.I.´s are below 3.


 Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with3HTdR, was within the range commonly recorded for animals of this strain and age.

The individual body weight values are included in Annex 1.

Lymph Node Weights

The measured lymph node weights of all animals treated were recorded after sacrifice. A relevant increase in lymph node weights was not observed in any of the test item treated groups in comparison to the vehicle control group.

Ear Weights

The measured ear weights of all animals treated were recorded after sacrifice. A relevant increase in ear weights was not observed in any of the test item treated groups in comparison to the vehicle control group.

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Results of the formulation analysis showed that all measured concentrations of Golpanol ALS wasserfrei were within the acceptable range set at 100 ± 15% of the nominal concentration.The obtained results ranged between 85 – 98.2% of the nominal values.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information