Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 17 March 2008 and 2 April 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Recently conducted GLP compliant study using the most recent test methods

Data source

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

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)
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): JKY-214
- Physical state: white solid
- Analytical purity: No information
- Lot/batch No.: Y002E
- Expiration date of the lot/batch: No information
- Stability under test conditions: No information. Assumed stable due to no data to the contrary
- Storage condition of test material: room temperature in the dark
- Other:

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Limited
- Age at study initiation: 8 - 12 weeks old
- Weight at study initiation: 15 - 23g
- Housing: housed in suspended solid-floor polypropylene cages
- Diet (e.g. ad libitum): Free access to Certified Rat and Mouse Diet
- Water (e.g. ad libitum): Free access to mains tap water
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06.00 to 18.00) and twelve hours darkness

IN-LIFE DATES: not reported

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10%, 5% or 2.5% w/w in dimethyl formamide
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: 10% w/w in dimethyl formamide
- Irritation: None. White residual test material upto 4 days after exposure
- Lymph node proliferation response: Not recorded

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Stimulation index
- Criteria used to consider a positive response: threefold or greater increase in stimulation index compared with control

TREATMENT PREPARATION AND ADMINISTRATION:

Test Material Administration
Groups of four mice were treated with the test material at concentrations of 10%, 5% or 2.5% 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 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.

iH-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 H-methyl thymidine (3HTdR: 80 nCi/ml, specific activity 2.0 Ci/mmol, GE Healthcare UK Ltd) giving a total of 20 μCi to each mouse.

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 P-scintillation counting. The "Poly Q™" 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 (Beckman Instruments Inc, Fullerton, CA, USA).


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

Results and discussion

Positive control results:
α-Hexylcinnamaldehyde, Tech, 85% was determined to be a sensitiser under the conditions of the test.

Concentration (% v/v) in
dimethyl formamide Stimulation Index Result
5 2.05 Negative
10 3.52 Positive
25 6.87 Positive

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Vehicle: n/a 2.5%: 1.12 5%: 0.87 10%: 1.26
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle: 6718.96 2.5%: 7545.18 5%: 5848.02 10%: 8432.72

Any other information on results incl. tables

Clinical Observations, Bodyweight and Mortality Data - Preliminary Screening Test

Concentration (% w/w) in dimethyl formamide

Animal Number

Bodyweight (g)

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-

Dose

Post Dose

Pre- Dose

Post Dose

Pre- Dose

Post Dose

10

S-l

22

20

0

0

0

ORt

0

ORt

ORt

0

0

0 = No signs of systemic toxicity

Rt = White residual material present on ears

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

Concentration (% w/w) in dimethyl formamide

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

6718.96

839.87

na

na

2.5

7545.18

943.15

1.12

Negative

5

5848.02

731.00

0.87

Negative

10

8432.73

1054.09

1.26

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 Individual Clinical Observations and Mortality Data

Concentration (% w/w) in dimethyl formamide

Animal

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Number

Pre- Dose

Post Dose

Pre- Dose

Post Dose

Pre- Dose

Post Dose

 

1-1

0

0

0

0

0

0

0

0

0

Vehicle

1-2

0

0

0

0

0

0

0

0

0

1-3

0

0

0

0

0

0

0

0

0

 

1-4

0

0

0

0

0

0

0

0

0

 

2-1

0

0

0

0

0

0

0

0

0

2.5

2-2

0

0

0

0

0

0

0

0

0

2-3

0

0

0

0

0

0

0

0

0

 

2-4

0

0

0

0

0

0

0

0

0

 

3-1

0

0

0

ORt

0

ORt

0

0

0

5

3-2

0

0

0

ORt

0

ORt

0

0

0

3-3

0

0

0

ORt

0

ORt

0

0

0

 

3-4

0

0

0

ORt

0

ORt

0

0

0

 

4-1

0

0

0

ORt

0

ORt

ORt

ORt

0

10

4-2

0

0

0

ORt

0

ORt

ORt

ORt

0

4-3

0

0

0

ORt

0

ORt

ORt

ORt

0

 

4-4

0

0

0

ORt

0

ORt

ORt

ORt

0

0 = No signs of systemic toxicity

Rt = White residual material present on ears Individual Bodyweights and Bodyweight Changes

Concentration (% w/w) in dimethyl formamide

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

20

21

1

1-2

18

19

1

1-3

19

20

1

1-4

21

21

0

2.5

2-1

18

20

2

2-2

19

20

1

2-3

18

21

3

2-4

20

21

1

5

3-1

18

19

1

3-2

19

20

1

3-3

18

20

2

3-4

19

20

1

10

4-1

19

20

1

4-2

19

20

1

4-3

20

22

2

4-4

20

21

1

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test sample is conisdered a non-sensitiser under the conditions of the test.
Executive summary:

The skin senstisation has been assessed by means of exposure to mice pre-treated with Thymidine according to EU method B429 in compliance with GLP. As the Stimulation Index of the test sample was below the Stimulation Index of the positive control, the test sample was considered to be non-sensitising.