Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 January 2014 to 25 February 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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:
solid
Details on test material:
- Appearance: Off-white solid
- Storage Conditions: room temperature in the dark, in a closed container under nitrogen

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Eight to twelve weeks of age
- Weight at study initiation: 15 to 23 g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes. The animals were provided with environmental enrichment items.
- Diet: ad libitum
- Water: ad libitum access to mains tap water
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: 30 to 70 % (relative)
- Air changes: The rate of air exchange was at least fifteen changes per hour
- Photoperiod: Lighting was controlled by a time switch to give twelve hours of continuous light (06:00 to 18:00) and twelve hours of darkness.

Study design: in vivo (LLNA)

Vehicle:
other: 1 % pluronic L92 in distilled water
Concentration:
1, 2.5 and 5 % w/w
No. of animals per dose:
5 females per dose
Details on study design:
RANGE FINDING TEST
Using available information regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using four mice, one mouse per test material concentration. The mice were treated by daily application of 25 µL of the test material at concentrations of 25, 10, 5 or 2.5 % w/w in 1 % pluronic L92 in distilled water, to the dorsal surface of each ear for up to three consecutive days (Days 1, 2 and 3). This vehicle was chosen as it produced the highest concentration that was suitable for dosing.
All mice were observed twice on Day 1, mice treated at concentrations of 10, 5 or 2.5 % w/w in 1 % pluronic L92 in distilled water were observed twice daily on Days 2 and 3 and the surviving mice treated at concentrations of 5 or 2.5 % w/w in 1 % pluronic L92 in distilled water were observed once on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and of the surviving mice on Day 6. The body weight of each mouse that was humanely killed was recorded immediately prior to termination.
Where necessary the thickness of each ear was measured 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.
The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the concentration of 5 % w/w.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION
Groups of five mice were treated with the test material at concentrations of 5, 2.5 or 1 % w/w in 1 % pluronic L92 in distilled water. 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.
The positive control animals were similarly treated; 25 µL of alpha-Hexylcinnamaldehyde (85 %) at a concentration of 25 % v/v in 1 % pluronic L92 in distilled water was applied to the dorsal surface of each ear.

³H-METHYL THYMIDINE ADMINISTRATION
Five days following the first topical application of the test material, vehicle control or positive control 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) 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.
- Body Weights: The body weight 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 g) for ten minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5 % Trichloroacetic acid (TCA).
- Determination of ³HTdR Incorporation: After approximately eighteen hours of incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, re-suspended 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 Q (TM)" 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).

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE
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 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 material (alpha-Hexylcinnamaldehyde) gave a Stimulation Index of 7.06 when tested at a concentration of 25 % v/v in 1 % pluronic L92 in distilled water, thus demonstrating the sensitivity and reliability of the test system.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
1 % w/w
Key result
Parameter:
SI
Value:
1.17
Test group / Remarks:
2.5 % w/w
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
5 % w/w
Parameter:
other: disintegrations per minute (DPM)
Value:
2 579.93
Test group / Remarks:
vehicle control
Parameter:
other: disintegrations per minute (DPM)
Value:
3 087.62
Test group / Remarks:
1 % w/w
Parameter:
other: disintegrations per minute (DPM)
Value:
3 017.48
Test group / Remarks:
2.5 % w/w
Parameter:
other: disintegrations per minute (DPM)
Value:
3 353.15
Test group / Remarks:
5 % w/w

Any other information on results incl. tables

Preliminary Screening Test

The animals treated with the test material at concentrations of 25 or 10 % w/w in 1 % pluronic L92 in distilled water were humanely killed, on Day 1 or Day 3, due to the occurrence of clinical signs of toxicity. Hunched posture and ptosis were noted in the animal treated with the test material at a concentration of 25 %. Increased activity and ptosis were noted in the animal treated with the test material at a concentration of 10 %. Body weight loss was noted in both of the animals that were humanely killed. Moderate to severe erythema and bleeding were noted on both ears of the animals that were humanely killed.

No signs of systemic toxicity or excessive irritation indicated by an equal to or greater than 25 % increase in mean ear thickness were noted in the animals treated with the test material at a concentration of 2.5 and 5 % w/w in 1 % pluronic L92 in distilled water; however very slight erythema was noted on both ears of the animal treated with the test material at a concentration of 5 % w/w in 1 % pluronic L92 in distilled water on Days 3 and 4.

 

Main Test

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

 

Table 1: Summary of Results

Treatment Group

Concentration (% w/w)

Mean dpm/Animal (±SD)

Stimulation Index

Result

Control

Vehiclex

2579.93 ± 1056.73

NA

NA

Test Material

1

3087.62 ± 827.83

1.20

Negative

2.5

3017.48 ± 1783.90

1.17

Negative

5

3353.15 ± 412.39

1.30

Negative

Positive Control

25

18216.07* ± 6160.42

7.06

Positive

SD = Standard deviation

x1 % pluronic L92 in distilled water

*Significantly different from the control group at p<0.01

Applicant's summary and conclusion

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material was determined to be a non-sensitiser.
Executive summary:

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 study was conducted in accordance with the standardised guidelines OECD 429 and EU Method B.42 under GLP conditions.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 5 % w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five 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, 2.5 or 1 % w/w. A further group of five animals was treated with the vehicle alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser alpha-hexylcinnamaldehyde at a concentration of 25 % v/v in 1 % pluronic L92 in distilled water.

The mean radioactive disintegrations per minute per animal were 2579.93, 3087.62, 3017.48 and 3353.15 for the vehicle control and 1, 2.5 and 5 % w/w dose groups respectively. The Stimulation Indices were 1.20, 1.17 and 1.30 for the 1, 2.5 and 5 % w/w dose groups respectively.

Under the conditions of this study, the test material was determined to be a non-sensitiser.