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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guidelinestudy (OECD 442B)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
(July 22, 2010)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Bioassay, Labor für biologische Analytik GmbH, Heidelberg, Germany)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(2-hydroxyethoxy)-3-[4-(2-methyloctyl)phenoxy]propan-2-ol; 2-(2-hydroxyethoxy)-3-[4-(2-methyloctyl)phenoxy]propan-1-ol
EC Number:
807-586-4
Cas Number:
634602-80-5
Molecular formula:
Unspecified
IUPAC Name:
1-(2-hydroxyethoxy)-3-[4-(2-methyloctyl)phenoxy]propan-2-ol; 2-(2-hydroxyethoxy)-3-[4-(2-methyloctyl)phenoxy]propan-1-ol

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: pretest 1: 9 weeks (beginning of treatment), pretest 2: 10 weeks (beginning of treatment), main test: 8 weeks (beginning of treatment)
- Weight at study initiation [g]: 19.8 g+/- 0.9 (vehicle), 20.0 +/- 1.2 (2.5%), 19.7 +/- 0.8 (5%), 20.0 +/- 1.2 (10%), 19.5 +/- 0.6 (25% HCA)
- Housing: single housing in Makrolon cages, type II
- Diet: STANRAB (P) SQC; SDS Special Diets Services, Altrip, Germany
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2.5, 5 and 10% (w/w)
No. of animals per dose:
5 (main test)
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 442B. The highest test item concentration, which could be technically used, was 100%.
- Irritation / Lymph node proliferation response: To determine the highest non-irritant test concentration that does not induce signs of systemic toxicity at the same time, two pre-tests were performed in twelve animals (2 animals per test item concentration and vehicle, each). Twelve mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 100, 50, 25 and 10% (w/w) and with the vehicle AOO once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible reddening of the ear skin. Furthermore, prior to the first application of the test item (day 0), on day 2 and before sacrifice (day 5) the ear thickness was determined using a micrometer (C110TKroeplin, 36381 Schluechtern, Germany). Additionally the ears were punched after sacrifice (day 5) at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm²) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if reddening of the ear skin of a score value ≥ 3 was observed at any observation time and/or if an increase in ear thickness of ≥ 25% was recorded on day 2 or day 5. The measured ear weight was also considered in this evaluation. In the pre-tests the highest concentration that could be technically used was 100% (w/w). Signs of systemic toxicity were not observed in the pre-tests. At the tested concentrations of 100 and 50% the animals showed signs of local irritation as confirmed by ear swelling measurements. Both animals in the 100% dose group and one of two animals in the 50% dose groups showed increased ear swellings > 25%. In dose group 25% the cut-off value of 5% body weight loss was exceeded in one animal. Test item residues and scaling were observed in the 100% and 50% dose group, a very slight erythema and scaling was noted in the 25% and 10% dose group. In the 10% dose group the measured values were below the cut-off values. Therefore, the highest concentration tested in the main study was 10%.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA: BrdU-ELISA
- Criteria used to consider a positive response: First, that exposure to at least one concentration of the test item resulted in an incorporation of BrdU at least 1.6-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: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 2.5, 5 and 10% (w/w) in AOO. The application volume 25 μL/ear/day was spread over the entire dorsal surface of each ear once daily for three consecutive days. The control test group of mice was treated with an equivalent volume of the relevant vehicle alone. The positive control test group of mice was treated with 25% α-hexyl cinnamaldehyde dissolved in acetone : olive oil (4:1 v/v). Four days after the first topical application (day 5) 0.5 mL of BrdU solution (5mg/per mouse/injection in DPBS (10 mg/mL)) were intraperitoneally injected. Approximately 24 hours after treatment with BrdU the mice were sacrificed and the draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal) and weighed immediately. After the lymph nodes were excised, the ear weights were determined. The incorporation of BrdU into lymph node cells was determined using a commercial cell proliferation assay kit (Roche Applied Science, Mannheim). Furthermore, the lymph node cell count was determined for each animal.
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, for the ear weights, the lymph node weights and lymph node cell count, and for the BrdU values. For the calculations excel (Version 2010) was applied. A statistical analysis was conducted on the BrdU values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group. For all statistical calculations Statistica (Version 10) was used. A Mann-Whitney-U test for non-parametric comparison was applied as statistical test. Statistical significance was set at the five percent level (p < 0.05). However, both biological and statistical significance were considered.
together.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Vehicle control: 1.0 (0.7, 1.1, 1.6, 1.1, 0.5); 2.5%: 1.7 (2.0, 1.1, 1.5, 3.2, 1.0); 5%: 1.3 (1.4, 0.5, 2.2, 1.8, 0.7); 10%: 2.1 (1.6, 2.0, 3.5, 2.4, 0.9); 25% HCA: 5.0 (6.6, 4.2, 4.0, 4.6, 5.5); EC1.6 (estimated concentration for a S.I. of 1.6): 6.9%; this value was calculated with the 5% and 10% dose group only, because the S.I. of the low dose group was assessed to be overrated. One animal of this dose group showed a BrdU labeling index more than twice as high as the other animals of this group. Without the BrdU value of this animal the S.I. of the low dose group would be 1.38 and clearly negative.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: not applicable

Any other information on results incl. tables

Viability / Mortality: In all groups, the animals did not show any relevant signs of systemic toxicity; no mortality was observed during the experimental period.

Clinical signs: The animals did not show any relevant signs of systemic toxicity. Signs of local skin reaction, observed in the high dose group on day 5, were incrustation in one animal and scaling in all animals.

Body weights: The body weight of the animals recorded prior to the first application and prior to sacrifice were within the range commonly recorded for animals of this strain and age.

Lymph Node Weights and Cell Counts: A statistically significant increase in lymph node weights and - cell counts was observed in the 10% group (weight: + 29%, cell count: + 41%) and the positive control (weight: + 139%, cell count: + 168%) in comparison to the vehicle control group. A statistically significant increase in lymph node cell counts was also observed in the 2.5% group (+ 18%) in comparison to the vehicle control group.

Ear weights: A statistically significant increase in ear weights was not observed in the test item groups, but in the positive control in comparison to the vehicle control group. According to OECD guideline 442B, an increase in ear weight exceeding the threshold

value of 25% was considered to be indicative for excessive local skin irritation. This threshold was not exceeded in any test item treated group.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information