Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 September 2012 - 9 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP 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.

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 lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Appearance: pale pink powder
- Storage conditions: Room temperature, in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
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: 18 - 21 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: 18 September 2012 To: 9 October 2012

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
25, 10 and 5 % w/w
No. of animals per dose:
4 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 at a concentration of 25 % w/w in propylene glycol, 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, 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 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 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. 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. 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 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.

INTERPRETATION OF RESULTS
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) 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):
other: Phenylacetaldehyde (CAS: 122-78-1) at 2.5 % v/v in propylene glycol.

Results and discussion

Positive control results:
The positive control was administered at 2.5 % v/v in propylene glycol and produced a stimulation index of 14.63. 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
Remarks on result:
other: 5 % w/w: 1.40 10 % w/w: 1.52 25 % w/w: 1.47 The result at all three concentrations was negative.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Disintegrations per minute and disintegration per minute per node are displayed in Table 1. The highest dpm was found in the 10 % w/w group, which had a dpm of 12324.68 (dpm/Node: 1540.59)

Any other information on results incl. tables

Table 1: Disintegrations per Minute (DPM), Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in propylene glycol

dpm

dpm/Node

Stimulation Index

Result

Vehicle

8125.66

1015.71

-

-

5

11338.53

1417.32

1.40

Negative

10

12324.68

1540.59

1.52

Negative

25

11921.33

1490.17

1.47

Negative

Positive Control

118861.70

14857.71

14.63

Positive

Disintegrations per Minute/Node obtained by dividing the disintegrations per minute value by 8 (the total number of lymph nodes).

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the study.

 

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 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.

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 four animals, were treated with 50 µL (25 µL per ear) of the test material as a suspension in propylene glycol at concentrations of 25, 10 and 5 % w/w. A further group of four animals was treated with propylene glycol alone. A concurrent positive control test, using a group of four animals, was performed with Phenylacetaldehyde, at a concentration of 2.5 % v/v in propylene glycol.

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.