Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
14 Apr - 16 May 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

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)
Version / remarks:
adopted in 2002
Deviations:
no
GLP compliance:
yes
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, C18-unsaturated, dimers, 2-ethylhexyl esters
- Physical state: clear amber liquid
- Analytical purity: 100%
- Lot/batch No.: 1548
- Expiration date of the lot/batch: 28 January 2011 (allocated by NOTOX, 1 year after receipt of the test substance)
- Storage condition of test material: RT in the dark
- Density: 0.906 g/mL

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Horst, The Netherlands
- Age at study initiation: 10 weeks
- Mean weight at study initiation: 20.9 g
- Housing: Individual housing in labelled Makrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment.
The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions, accommodation was as described above except that the animals were group housed in Makrolon cages (MIII type; height 18 cm)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 (actual range: 19.7 – 22.9)
- Humidity (%): 40-70 (actual range: 37 - 83)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25, 50 and 100% (w/w)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation at the highest concentration. Based on the results, the highest test substance concentration selected for the main study was a 100% concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Disintegrations Per Minute (DPM) values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM. The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures.


TREATMENT PREPARATION AND ADMINISTRATION: The dorsal surface of both ears was epidermally treated (25 μL/ear) with the test substance concentration, at approximately the same time per day. Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After approximately five hours, all animals were killed by intraperitoneal injection with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS and radioactive measurements were performed using a Packard scintillation counter (2800TR).

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The six-month reliability check with alpha-hexylcinnamic aldehyde indicated that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI values calculated for the substance concentrations 25, 50 and 100% were 1.6, 1.6 and 2.5, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 1644, 1717 and 2675 DPM respectively. The mean DPM/animal value for the vehicle control group was 1060 DPM.

Any other information on results incl. tables

Table 1: Radioactivity measurements (individual animals)

group

 test substance1(% w/w)

animal

DPM / animal

 

 

 

 

1

0%

1

1529

 

 

(Vehicle)

2

578

 

 

 

3

1355

 

 

 

4

939

 

 

 

5

898

 

 

 

 

 

 

2

25%

6

678

 

 

 

7

2490

 

 

 

8

2104

 

 

 

9

1235

 

 

 

10

1712

 

 

 

 

 

 

3

50%

11

1506

 

 

 

12

1544

 

 

 

13

2260

 

 

 

14

1497

 

 

 

15

1780

 

 

 

 

 

 

4

100%

16

3393

 

 

 

17

2354

 

 

 

18

2537

 

 

 

19

2339

 

 

 

20

2751

 

 

 

 

 

1      Vehicle: Acetone/Olive oil (4:1 v/v).

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