Registration Dossier

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 - Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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)
Remarks:
Behörde Soziales, Familie, Gesundheit und Verbraucherschutz, Hamburg, Germany
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): Fatty acids, C8-10, mixed esters with adipic acid and trimethylolpropane
- Physical state: light yellowish, clear liquid
- Analytical purity: 100%
- Lot/batch No.: OE10428
- Storage condition of test material: at room temperature, in a tightly closed container; kept away from heat, sparks, flames and direct sunlight

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 62 days
- Weight at study initiation: 26-31 g
- Housing: before application, animals were housed in groups in MAKROLON cages (type III) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 15 cm. After application, animals were housed singly in order to prevent licking off the test item from the ears of the other animals. Granulated textured wood (Granulat A2, J. Brandenburg, Goldenstedt, Germany) was used as bedding material for the cages.
- Diet: commercial diet ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Air changes (per hr): 12-18
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Sep 2012 To: 27 Sep 2012

Study design: in vivo (LLNA)

Vehicle:
other: acetone/olive oil (3+1 v/v)
Concentration:
25, 50 and 100% (w/w)
No. of animals per dose:
6
Details on study design:
RANGE FINDING TESTS: a preliminary experiment was performed in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Three concentrations of 25 and 50% diluted with acetone/olive oil (3+1 v/v) and the undiluted test substance were applied to both ears of each animal on 3 consecutive days. Lymph node weights, body weights, the diameter of punch biopsies of both ears and ear thickness were determined.
- Irritation: no pronounced irritating properties were observed in this preliminary experiment at concentrations of 25%, 50% or 100%, and no differences in ear weight and ear thickness were noted.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: cell proliferation determined by lymph node cell count and lymph node weight
- Criteria used to consider a positive response: a stimulation index for the lymph node cell count above 1.4 was considered positive. For lymph node weight, significance at p ≤ 0.01 was considered positive (U-test according to MANN and WHITNEY). A possible concentration-response relationship for the lymph node weight in order to determine a possible sensitising potential was examined by linear regression analysis employing PEARSON's correlation coefficient. Outliers were determined according to the Nalimov test. Skin irritation was considered positive, if the value for the stimulation index of ear weight was above 1.1.

TREATMENT PREPARATION AND ADMINISTRATION: On Day 1 of the study, the weight of each animal was individually identified and recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer. Open application of 25 µL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear. The application procedure carried out on Day 1 was repeated on two consecutive days. On day 4, ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer. At sacrifice, body weights were determined and punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance. Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes of each animal were then stored on ice in PBS /0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean values and standard deviations of lymph node cell counts, lymph node weights and body weights were determined. For the diameter of punch ear biopsies and ear thickness, mean values and relative standard derivation were calculated. The determination of a positive result based on the lymph node weight was determined by statistical analysis using the U-test according to MANN and WHITNEY. A possible concentration-response relationship for the lymph node weight in order to determine a possible sensitising potential was examined by linear regression analysis employing PEARSON's correlation coefficient. Outliers were determined according to the Nalimov test.

Results and discussion

Positive control results:
The positive control hexyl cinnamic aldehyde induced a statistically significant (p ≤ 0.01) increase in the lymph node cell count (stimulation index: 2.035) and lymph node weight (stimulation index: 1.712). Based on these results, the study can be regarded as valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: - Lymph node cell count: 1.029, 1.308 and 1.212 at 25, 50 and 100%, respectively - Lymph node weight: 0.962, 1.173 and 1.135 at 25, 50 and 100%, respectively
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: not applicable

Any other information on results incl. tables

SKIN IRRITATION

The acute inflammatory skin reaction was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on Day 1 and Day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.

 

Stimulation indices – test substance:

- Ear weight: 1.022, 1.017 and 1.067 at 25, 50 and 100%, respectively

- Difference of ear thickness: 0.992, 1.042 and 1.029 at 25, 50 and 100%, respectively

 

Stimulation index – positive control (25%):

- Ear weight: 0.994

- Difference of ear thickness: 1.113

The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted.

BODY WEIGHT

The animal body weight was not affected by the treatment.

CLINICAL SIGNS

No signs of local or systemic intolerance were recorded.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: not classified
DSD: not classified