Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Report Date:
2011
Reference Type:
other: Amendment
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Laromer PO 33 F
- Physical state: yellowish liquid
- Analytical purity: The test item is a complex mixture of isomers and homologues components, so no purity can be stated (for details see analytical report BASF Study No.: 11L00272)
- Purity test date: 2011-12-19
- Lot/batch No.: 110007P040
- Expiration date of the lot/batch: 2012-07-07
- Stability under test conditions: not indicated, concentrations will be checked by the sponsor
- Storage condition of test material: room temperature below 25°C
- other: Substance no. 11/0457-1

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd (2nd pre-test); CBA/CaCrl (1st, 3rd pre-test and main study)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CBA/CaOlaHsd: Harlan Laboratories, Horst, Netherlands; CBA/CaCrl: Charles River UK
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 17.4 - 21.6g
- Housing: groups in Makrolon Type II (pretest) or Type III (main study) cages
- Diet (e.g. ad libitum): Pelleted standard diet ad lib. (Harlan laboratories, Horst, Netherlands)
- Water (e.g. ad libitum): tap water ad lib.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C
- Humidity (%):45-65% (main study)
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0%, 1.19%, 2,94%, 6.0% (w/w) (main study - concentration was corrected according to sample analysis)
50%, 100% (1st pre-test)
10%, 25% (2nd pre-test)
2.5%, 5% (3rd pre-test)
No. of animals per dose:
5 (main study)
2 (pre-tests)
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: soluble in aceton:olive oil (4:1 v/v)
- Irritation: considered excessive, if erythema ≥3 at any time point or increase in ear thickness ≥ 25% on day 3 or 6
50% / 100% (1st pretest): erythema up to grade 2 and ear swelling of 51% and 81%.
10%/25% (2nd pretest): erythema up to grade 2 and ear swelling of 33.3% and 102.1%
2.5%, 5% (3rd pretest): erythema grade 1 (day4-6), only slight ear swelling (app. 8%)
- Lymph node proliferation response: not measured in pretests

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: SI value ≥ 3 and dose response relationship is observed, though local effects or immunological suppression must also be considered.

TREATMENT PREPARATION AND ADMINISTRATION:
25µl of each concentration was spread evenly onto the dorsal suface of each ear on three consecutive days.
5 days after the first application 250µl of phosphate-buffered saline (PBS) containing 20.0 µCi of ³HTdR (equivalent to approximately 79.9 µCi/mL ³HTdR) were injected into each test and control mouse via the tail vein. Mice were euthanised 5 hours later.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test was used for identification of possible outliers.

Results and discussion

Positive control results:
SI (10%) = 2.18
SI (25%) = 8.08
EC3 = 12.1%
historical control data: SI (25%) = 5.04 - 10.03

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: SI (1.19%): 0.88 SI (2.94%) : 0.87 SI (6%): 1.84
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: control: 1173.5 ± 399.2 1.19%: 1030.5 ± 498.4 2.94%: 1019.5 ± 124.7 6%: 2163.3 ± 644.9

Any other information on results incl. tables

No local irritation was observed.

Applicant's summary and conclusion

Conclusions:
The test item was thus not a skin sensitiser under the test conditions of this study.
Executive summary:

In this study the test item dissolved in acetone:olive oil (4+1 (v/v)) was assessed for its skin sensitizing potential using the Local Lymph Node Assay (LLNA) in mice using test item concentrations of 1.19, 2.94, and 6% (w/w).The highest concentration tested was the highest concentration that could be applied whilst avoiding excessive local skin irritation (as determined by three pre-experiments).

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. A statistically significant increase in ear weights was observed in the high dose group in comparison to the vehicle control group (p >0.05). However, this was considered to be not biologically relevant, as the mean value of the group was still within the range of historical vehicle control data for the ear weight. Furthermore, the cutoff-value for a positive response regarding the ear weight index of 1.1 reported for BALB/c mice was not exceeded in any dose group. In this study Stimulation Indices (S.I.) of 0.88, 0.87, and 1.84 were determined with the test item at concentrations of 1.19, 2.94, and 6% (w/w) test item in acetone:olive oil (4+1 (v/v)), respectively. A clear dose response was not observed. A statistically significant increase in DPM value and in lymph node cell count was determined in the high dose group in comparison to the vehicle control group (p > 0.05). Still, as the Stimulation Index determined for this group was well below the threshold value of 3 for a positive response, this was not biologically relevant. Also, the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was not exceeded in any dose group. The lymph node weights did not show a statistically significant increase in any dose group in comparison to the vehicle control group. An outlier was identified in the low dose group (DPM value determined for animal number 7). However, as the corresponding lymph node weight value confirmed the result of the DPM value and as exclusion of the outlier did not change the overall test result, the value in question was not excluded from calculation.