Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 18 July 2007 and 24 July 2007.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

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 lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification: C-Y9
Description : dark yellow/orange powder
Batch number : MC-21
Date received : 11 June 2007
Storage conditions: room temperature in the dark over silica gel

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Female CBA/Ca (CBA/CaBkl) strain mice were supplied by B & K Universal Ltd, Hull, UK
- Age at study initiation: Eight to twelve weeks old.
- Weight at study initiation: 15 to 23 g
- Housing: The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): Free access to food (Certified Rat and Mouse Diet) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains tap water was allowed throughout the study.
- Acclimation period: At least fice days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): The rate of air exchange was approximately fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:
other: 1% pluronic L92 in distilled water.
Concentration:
Test material at concentrations of 5%, 10% or 25% w/w in 1% pluronic L92 in distilled water.
No. of animals per dose:
Groups of 4 mice per dose.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: For the purpose of the study, the test material was freshly prepared in 1% pluronic L92 in distilled water. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.
- Irritation: Information available suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration.


MAIN STUDY

TREATMENT PREPARATION AND ADMINISTRATION:
Test material administration:
The mice were treated by daily application of 25 µl 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.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol, GE Healthcare UK Ltd) giving a total of 20 µCi to each mouse.

Observations:
Clinical observations:
All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.

Bodyweights:
The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures:
Termination:
Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 ml of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension:
A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by beta-scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system.
















Positive control substance(s):
other: 2,4 Dinitrobenzenesulfonic acid, sodium salt

Results and discussion

Positive control results:
Three groups, each of five animals, were treated with 50 µl (25 µl per ear) of 2,4 Dinitrobenzenesulfonic acid, sodium salt as a solution in 1% pluronic L92 in distilled water at concentrations of 1%, 10% and 20% w/w. A further control group of five animals was treated with 1% pluronic L92 solution in distilled water alone.

Results:
. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% w/w) in 1% pluronic L92 in distilled water: 1%
Stimulation Index: 1.39
Result: Negative

Concentration (% w/w) in 1% pluronic L92 in distilled water: 10%
Stimulation Index: 11.33
Result: Positive

Concentration (% w/w) in 1% pluronic L92 in distilled water: 20%
Stimulation Index: 19.34
Result: Positive

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 1. A stimulation index of less than 3 was recorded for the three concentrations of the test material (5%, 10% and 25% w/w in 1% pluronic L92 in distilled water).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 1.

Any other information on results incl. tables

Estimation of the Proliferative Response of Lymph Node Cells

Table1               Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration

(% w/w) in
1% pluronic L92 in distilled water

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

4901.72

612.72

na

na

5

3638.07

454.76

0.74

Negative

10

5498.48

687.31

1.12

Negative

25

6911.60

863.95

1.41

Negative


dpm=    Disintegrations per minute

a=          Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b=         Stimulation Index of 3.0 or greater indicates a positive result

na =      Not applicable

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Orange-coloured staining on the ears was noted post-dose on Days 1 and 2 in animals treated with the test material at a concentration of 25% w/w in 1% pluronic L92 in distilled water.

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 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. 

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

§        OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted)

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

Methods. 

Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test material as a solution in 1% pluronic L92 in distilled water at concentrations of 5%, 10% or 25% w/w. A further group of four animals was treated with 1% pluronic L92 in distilled water alone.

Results. 

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (% w/w) in
1% pluronic L92 in distilled water

Stimulation Index

Result

5

0.74

Negative

10

1.12

Negative

25

1.41

Negative

Conclusion. 

The test material was considered to be a non‑sensitiser under the conditions of the test.