Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 February - 03 March 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was performed according to OECD and EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2002)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(2004)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
(2003)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Y-513
- Expiration date of the lot/batch: 19 February 2013
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark
- pH (1% in water): 7.6 - 7.0

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle Cedex, France
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: 19-24 g
- Housing: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material. Paper was supplied as cage-enrichment.
- Diet: Free access to pelleted rodent diet.
- Water: Free access to tap water.
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 - 23.6
- Humidity (%): 37 - 74
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN LIFE PHASE: 18 february 2008 To: 03 March 2008

Study design: in vivo (LLNA)

Vehicle:
methyl ethyl ketone
Concentration:
5%, 10% or 25%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
A series of two test substance concentrations was tested: 50% and 25%. The highest concentration, selected from this series, was the maximum concentration that could technically be applied.
The test system, procedures and techniques were identical to those used during days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (8-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the ear was cleaned of residual test substance with tap water and the irritation was assessed. Bodyweights were determined on day 3. The animals were sacrificed after the final observation and no necropsy was performed.

Yellow staining by the test substance prevented scoring of erythema. Since the 50% concentration did not remain adhered to the ear, a 25% concentration was selected as the highest concentration that could be used in the main study.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: 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 sensitiser, based on the test guideline and recommendations done by ICCVAM


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. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing.
The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine.
After approximately five hours, all animals were killed by intraperitoneal injection with pentobarbital Euthesate® (0.2 mL/animal). 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
none

Results and discussion

Positive control results:
The SI values calculated for the positive control at concentrations 5, 10 and 25% were 1.0, 2.0 and 5.7 respectively. An EC3 value of 14.1% was calculated using linear interpolation.

The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 monthly HCA reliability checks of the recent years were 10.3, 9.5, 13.1 and 15.6%.

Based on the results, it was concluded 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 5, 10 and 25% were 1.1, 1.4 and 1.3 respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 419, 523 and 496 respectively. The mean DPM/animal value for the vehicle control group was 369.

Any other information on results incl. tables

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study. Yellow staining or test substance remnants prevented scoring for erythema. No oedema was observed in any of the animals examined. No substance-related changes in body weight gain were noted.

All nodes of the experimental and control animals were considered normal in size. No macroscopic abnormalities of the surrounding areas were noted.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Based on the results of an LLNA, performed according to OECD and GLP guidelines, Y-513 is found to be not a skin sensitizer.
Executive summary:

An LLNA was performed according to OECD and GLP guidelines with Y-513 administered at 5%, 10% and 25%. No mortality occurred and no symptoms of systemic toxicity were observed. Yellow staining or test substance remnants prevented scoring for erythema. No oedema was observed in any of the animals examined. No substance-related changes in body weight gain were noted.

All nodes of the experimental and control animals were considered normal in size. No macroscopic abnormalities of the surrounding areas were noted. Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10 and 25% were 419, 523 and 496 respectively. The mean DPM/animal value for the vehicle control group was 369. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.1, 1.4 and 1.3 respectively. Since there was no indication that the test substance elicited an SI > 3 when tested up to and including 25%, the test substance was considered to be not a skin sensitizer and is not classified according to Regulation (EC) 1272/2008.