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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-11-02 to 2009-11-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester. Oxon, UK
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: Individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Free access to 2014 Teklad Global Rodent diet (Harlan Teklad, Bicester, Oxon, UK)
- Water: Free access to mains tap water
- Acclimation period: 5 days minimum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light): Twelve hour cycle (06:00 to 18:00 continuous light).
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
50, 25 and 10 % w/w
No. of animals per dose:
4 animals per dose in the main test, with one animal in the preliminary test.
Details on study design:
RANGE FINDING TESTS
- Irritation and toxicity: A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the test material at a concentration of 50 % w/w in acetone/olive oil 4:1 to the dorsal surface of each ear for three consecutive days. The mouse was observed daily on days 1, 2 and 3 and once a day on days 4, 5 and 6. Signs of toxicity or excessive local irritation were recorded. The bodyweight of the mouse was recorded on day 1 (prior to dosing) and 6.
- Compound solubility: The vehicle was chosen based on its ability to produce the highest concentration that was suitable for dosing. The concentration for dosing was selected on the basis that the dose produces a solution or fine homogenous suspension that can be administered via a micropipette.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporations will be classified as a non-sensitiser.

TREATMENT PREPARATION AND ADMINISTRATION: Groups of four mice were treated with the test material at concentrations of 50 %, 25 % or 10 % in acetone/olive oil 4:1. The mice were treated daily by application of 25 µL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (1, 2, and 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 groups of four mice received the vehicle alone in the same manner.

Five days following the first topical application of the appropriate treatment 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) giving a total of 20 µCi to each mouse.

Five hours following 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.

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).

After 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase Trisafe). 3HTdR incorporation was measured by β-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 to reduce the risk of luminescence. After 20 minutes the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measure using the Beckman LS6500 scintillation system.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The stimulation Index was calculated by dividing the mean radioactive incorporation for each treatment group by the mean radioactive incorporation of the vehicle control.
Positive control results:
The Stimulation Index for α-Hexylcinnamaldehyde, Tech. 85 % was considered to be a positive sensitiser under the conditions of the test.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Please refer to table 5 under section 'Remarks on results including tables and figures for tabulated data' .
Parameter:
SI
Value:
2.34
Variability:
Not specified
Test group / Remarks:
10 % w/w in acetone/olive oil 4:1
Remarks on result:
other: Negative
Parameter:
SI
Value:
1.88
Variability:
Not specified
Test group / Remarks:
25 % w/w in acetone/olive oil 4:1
Remarks on result:
other: Negative
Parameter:
SI
Value:
1.41
Variability:
Not specified
Test group / Remarks:
50 % w/w in acetone/olive oil 4:1
Remarks on result:
other: Negative

Table 3: 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 acetone/olive oil 4:1

Stimulation Index

Result

10

2.34

Negative

25

1.88

Negative

50

1.41

Negative

 

Ta ble 4: Clinical observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration (% w/w) in acetone/olive oil 4:1

Animal Number

Bodyweight (g)

Day

1

2

3

4

5

6

Day

1

Day

6

Pre-Dose

Pre-Dose

Pre-Dose

Pre-Dose

Pre-Dose

Pre-Dose

50

S-1

20

20

0

0

0

0Fs

0

0Fs

0

0

0

 

0 = No signs of systemic toxicity

Fs = Brown coloured staining on the ears

Table 5: Disintegrations per minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in acetone/olive oil 4:1

dpm

Dpm/Nodea

Stimulation Indexb

Result

Vehicle

5807.79

725.97

na

na

10

13574.38

1696.80

2.34

Negative

25

10918.43

1364.80

1.88

Negative

50

8216.87

1027.11

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

 

Table 6: Individual Bodyweights and Bodyweight changes 

Concentration (% w/w) in acetone/olive oil 4:1

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

 

Vehicle

1-1

20

19

-1

1-2

17

18

1

1-3

18

19

1

1-4

19

19

0

10

2-1

20

20

0

2-2

18

18

0

2-3

17

17

1

2-4

19

20

0

25

3-1

19

19

0

3-2

21

21

0

3-3

19

19

0

3-4

19

20

1

 

50

4-1

21

19

-2

4-2

18

18

0

4-3

18

19

1

4-4

17

18

1

 

 

Interpretation of results:
other: Not classified according to EU criteria.
Conclusions:
Under the conditions of the study the test material was concluded to be a non sensitiser.
Executive summary:

The skin sensitisation potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 429 and EU Method B.42.

Under the conditions of the study the test material was concluded to be a non sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitisation potential of the test material was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 429 and EU Method B.42.

Under the conditions of the study the test material was concluded to be a non sensitiser.


Justification for selection of skin sensitisation endpoint:
Only one study is available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to sensitisation.

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance does not require classification with respect to sensitisation.