Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study test phase 2005-09-12 to 2005-09-27. Report completed and signed off 2005-12-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Meets criteria for classification as reliable without restriction according to Klimisch et al (1997)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identification: FSM-003B (S196439)
Description: black powder
Batch number: CE-501-2

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca (CBA/CaBkl) strain mice were supplied by B & K Universal Ltd, Hull, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.

The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (Certified Rat and Mouse Diet (Code 5LF2) supplied by BCM IPS Limited, London, UK) was allowed throughout the study.

The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0, 5, 10 and 25% w/w
No. of animals per dose:
four per dose group
Details on study design:
Following a preliminary screening test groups of four mice were treated with the test material at concentrations of 5%, 10% or 25% w/w in dimethyl formamide. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 uI of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.

Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250ul of phosphate buffered saline (PBS) containing 3H-methyl thymidine giving a total of 20 uCi to each mouse. Five hours following the administartion of 3HTdR all mice were killed and the draining auricular lymph nodes from the mice were excised and pooled for each experimental group. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation. The pooled lymph node cells were pelleted and resuspended. To precipitate out the radioactive material the pellet was resuspended in Trichloroacetic acid (TCA). After approx. 18h incubation the precipitates were recovered by centrifugation and transferred to scintillation fluid. HTdR incorporation was measured by B-scintillation counting. Following a period of time in darkness, the number of radioactive disintegrations per minute was then measured.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None used.

Results and discussion

Positive control results:
The positive control responded as expected and confirmed that the test system was operating as expected.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 5 % = 1.54 10% = 1.71 25% = 2.41
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle = 5670.65 5% = 8729.5 10% = 9678.43 25% = 13687.24

Any other information on results incl. tables

Clinical observations and Mortality Data

There were no deaths during this study. No signs of systemic toxicity were noted in the test or control animals during the test. Black staining of the fur and ears was noted one hour post dosing on Days 1, 2 and 3.

Bodyweight

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information not classified Criteria used for interpretation of results: EU
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of the test.
Executive summary:

Introduction

This study was undertaken to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical administration to the dorsal surface of the ear. The method was designed to meet the requirements of the following guidelines:

OECD Guidelines for the Testing of Chemicals 429 'Skin Sensitisation: Local Lymph Node Assay' (adopted 24 April 2002)

Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC

Results and Conclusion

All three test concentrations (5% w/w, 10% w/w, 25% w/w) gave a negative result. The test material was considered to be a non-sensitiser under the conditions of the test.