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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June 2012 to 28 June 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/Ca (CBA/CaOlaHsd)
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: the animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum access to mains tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25°C
- Humidity (%): 30 - 70%
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): lighting was controlled by a time switch to give twelve hours continuous light (0600 to 1800) and twelve hours darkness.

IN-LIFE DATES: From: 14 June 2012 To: 28 June 2012

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100 (undiluted test material), 50 and 25 % v/v
No. of animals per dose:
5 animals per dose
Details on study design:
PRELIMINARY SCREENING TEST
A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

MAIN TEST
- 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 five mice received the vehicle alone in the same manner.

- 3H-Methyl Thymidine Administration
Five days following the first topical application of the test material or vehicle (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) 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 followed by cervical separation. For each individual animal of each group, the draining auricular lymph nodes were excised and processed. For each individual animal, 1 mL of PBS was added to the lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal 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. The lymph node cells 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 lymph node cells were pelleted at 1400 rpm (approximately 190 x gravity) 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 x gravity) for ten 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. 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).

INTERPRETATION OF RESULTS
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

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 incorporation will be classified as a "non-sensitiser".
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett's Multiple Comparison test was used and for non-homogenous datasets Dunnett's T3 Multiple Comparison Method was used.

Results and discussion

Positive control results:
The positive control was administered at 25% v/v in acetone/olive oil 4:1 and produced a stimulation index of 5.76. This corresponds to a positive result.
Therefore, the positive control was found to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.66
Test group / Remarks:
25% v/v
Parameter:
SI
Value:
2.2
Test group / Remarks:
50% v/v
Parameter:
SI
Value:
1.96
Test group / Remarks:
100% v/v
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Disintegrations per minute are displayed in Table 1. The highest dpm was found in the 50 % v/v group, which had a dpm of 7060.18.
Cellular proliferation data / Observations:
- Preliminary Screening Test
No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Based on this information the undiluted test material was dosed at 50% and 25% v/v in acetone/olive oil 4:1 test concentrations in the main study.

- Main Test
None of the animals died during the study and no signs of systemic toxicity were noted in either test or control animals. Bodyweight changes in the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.
Disintegrations per minute and stimulation index data provided in Table 1.

Any other information on results incl. tables

Table 1 Individual Disintegrations per Minute and Stimulation Indices

Concentration (% v/v) in Acetone/Olive Oil 4:1

Animal Number

dpm/Animal

Mean dpm/Animal (Standard Deviation)

Stimulation Index

Result

 

 

Vehicle

1-1

2844.07

 

 

3215.81

(± 1053.38)

 

 

-

 

 

-

1-2

2487.28

1-3

4167.09

1-4

2095.85

1-5

4484.76

 

 

25

2-1

5160.23

 

 

5343.08*

(± 342.36)

 

 

1.66

 

 

Negative

2-2

5027.72

2-3

5834.42

2-4

5129.12

2-5

5563.90

 

 

50

3-1

7561.57

 

 

7060.18***

(± 1486.09)

 

 

2.20

 

 

Negative

3-2

9262.66

3-3

6185.95

3-4

6949.54

3-5

5341.17

 

 

100

4-1

6624.77

 

 

6302.47**

(± 1243.43)

 

 

1.96

 

 

Negative

4-2

6495.92

4-3

7974.24

4-4

5862.80

4-5

4554.61

†Total number of lymph nodes per animal is 2

* Significantly different from the control group (p < 0.05)

** Significantly different from the control group (p < 0.01)

*** Significantly different from the control group (p < 0.001)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of this study in accordance with EU criteria.
Executive summary:

The potential of the test material to act as a sensitiser was investigated in accordance with the standardised guidelines OECD 429 and EU Method B.42 under GLP conditions.

The study assessed the skin sensitisation potential of the test material in female mice of the CBA/Ca strain following topical application to the dorsal surface of the ear. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the undiluted test material or the test material as a solution in acetone/olive oil 4:1 at concentrations of 50 or 25%. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was performed with α-hexylcinnamaldehyde at a concentration of 25 % v/v in acetone/olive oil 4:1.

The results for all three concentrations of the test material were negative, whilst the positive control material gave the expected result.

Therefore, the test material was considered to be a non-sensitiser under the conditions of this study in accordance with EU criteria.