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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:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with detailed documentation.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
-reliability scoring based on 2002 guideline
Deviations:
yes
Remarks:
During the acclimation period, relative humidity in the animal room was between approximately 20-65% for a maximum of 15 hours.
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
During the acclimation period, relative humidity in the animal room was between approximately 20-65% for a maximum of 15 hours.
GLP compliance:
yes (incl. QA statement)
Remarks:
Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): diisopropyl ether
- Analytical purity: 99.5%
- Lot/batch No.: 09062392
- Expiration date of the lot/batch: June 2010
- Stability under test conditions: Not reported
- 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: 17.6 to 22.1 g
- Housing: Single caging in Makrolon Type II with wire mesh top and granulated soft wood bedding
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (from Harlan Laboratories B.V., 5960 AD Horst/The 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 the dosing under test conditions


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 ºC
- Humidity (%): 20-65%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12


Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50, or 100%
No. of animals per dose:
4 females per group
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which can be technically used was 100% of the undiluted test item.
- Irritation: To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 50 and 100% each on three consecutive days. Clinical signs were recorded within 1 hour and 24 ± 4 hours after each application, as well as on Day 7. At the tested concentrations, the animals did not show any signs of irritation or systemic toxicity.
- Lymph node proliferation response: Not reported.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: a test item is regarded as a sensitizer if (1) the exposure to at least one concentration of the test item resulted in an incorporation of 3-Hmethyl thymidine (3HTdR) at least 3-fold or greater than that recorded in control mice, as indication by the stimulation index, and (2) that the data are comparable with a conventional dose response


TREATMENT PREPARATION AND ADMINISTRATION:

Topical Application - Each test group of mice was treated by a topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 25, 50, or 100% in acetone:olive oil (4:1). The application volume, 25 µl, was spread over the entire dorsal surface of each ear once daily for three consecutive days. A further group was treated with the vehicle alone.

Five days after the first topical application, all mice were administered with 250 µl of 78.3 microCi/ml 3HTdR (corresponds to 19.6 µCi 3HTdR per mouse) by intravenous injection via a tail vein. Approximately 5 hours after treatment with 3HTdR all mice were euthanized buy intraperitonal injection of Pentobarbial-Natrium. The draining lymph nodes were excised and pooled. Single-cell suspensions were prepared and washed with phosphate buffered saline twice and then suspended in 5% tricholoracetic acid. Then the cells were incubated at approximately 4 ºC for 18 hours and the precipatates were resuspended in 5% tricholoracetic acid and transfered to plastic scintillation vials and mixed. The level of 3HtdR incorporation was measured on a beta-scintillation counter as disintegrations per minute (DPM).
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.

Results and discussion

Positive control results:
See attached Annex II of study report for positive control results

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 25% group - 0.75 50% group - 0.71 100% group - 0.80
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Control group - 5932 25% group - 4442 50% group - 4242 100% group - 4753

Any other information on results incl. tables

No deaths occurred during the study period. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. The body weights of animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study, Stimulation Indices of 0.75, 0.71, and 0.80 were determined with the test item at concentrations of 25, 50, and 100% in acetone:olive oil (4 +1), respectively. The test item diisopropyl ether was not a skin sensitizer under the test conditions.