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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:
From April 10 to June 12, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test conducted according to internationally accepted Guideline and in according to the GLP Principles
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/JN
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italy S.r.l.
- Age at study initiation: 8 - 12 Weeks at the time of treatment
- Weight at study initiation:
- Housing: The animals have been housed ub a limited access rodent facility, individually during the test.
- Cages: Bottomed in polysulphone solid. Measure 35.5x23.5x19 cm
- Nesting material: provided inside suitable bedding bags and changed at least twice a week
- Diet (e.g. ad libitum): ad libitum(laboratory rodent diet 4RF21, Mucedola S.r.l.)
- Water (e.g. ad libitum): ad libitum by water bottle
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 55 ± 15 %
- Air changes (per hr): aproximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark per day
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
50 %, 25 %, 10 %, 5 %, 2.5 %, 1 %, 0.5 %, 0.25 %, 0.1 %, 0.05 %
No. of animals per dose:
2 animals per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility:
A solubility trial was performed in order to establish if acetone/olive oil 4:1 v/v could be used as a vehicle. At concentrations of 50 and 25 % w/w in acetone/olive oil 4:1 v/v, formulations with particles in suspension were obtained

PRELIMINARY TEST
- DOSING
Frequency of treatment
The animals were treated for three consecutive days (Days 1, 2, 3) with the vehicle or test item formulations depending on the animal.

Dosing method
A dose volume of 25 μl/ear/day of the appropriate concentration was applied to the dorsal surface of each ear (50 μl/animal/day), using a micropipette

- IN LIFE OBSERVATIONS
Mortality and morbidity
Throughout the study all animals were checked twice daily.

Clinical signs
The animals were observed for clinical signs on:
Day 1: before and 1 hour after dosing.
Day 2 to 6: daily (approximately 1 hour after daily dosing, when applicable).

Body weight
The animals were weighed at allocation (Day 1) and on sacrifice (Day 6).

- TERMINATION
Euthanasia method and ear punch weight
The animals were sacrificed on Day 6 by carbon dioxide narcosis. After sacrifice, regularly shaped biopsies were obtained from both ears (ear punch) and weighed together. No necropsy was performed on the animals.

- IRRITATION:
Irritation to the skin was assigned a numerical value according to the value below
No erythema: 0
Very slight erythema: 1
Well defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema: 4
In the main assay, the test item was used at the higher concentrations that were systemically tolerated and judged not to cause excessive local skin irritation.
In particular, concentrations are considered irritant if:
– erythema grading (score) is ≥ 3 at any day of measurement and/or
– ear thickness is ≥ 25 % with respect to Day 1 and/or
– ear punch weight is ≥ 25 % with reference to the negative control group

Ear thickness measurement
The ear thickness was measured by a suitable micrometer on Day 1 (before dosing), on Day 3 (before dosing) and on Day 6

MAIN STUDY
- DOSING WITH THE TEST OR CONTROL ITEMS
Frequency of treatment
The animals were treated for three consecutive days (Days 1, 2, 3) with the vehicle, test or control item formulations.

Dosing method
A dose volume of 25 μl/ear/day of each selected concentration and controls was applied to the dorsal surface of each ear (50 μl/animal/day), using a micropipette.
5-bromo-2-deoxyuridine (BrdU) treatment
The animals were treated intraperitoneally, on Day 5 (once only), with 0.5 ml/animal of a solution of BrdU at a concentration of 10 mg/ml in physiological saline (0.9 % NaCl), using a plastic graded syringe.

- IN LIFE OBSERVATIONS
Mortality and morbidity
Throughout the study all animals were checked twice daily.

Clinical signs
The animals were observed for clinical signs before dosing commenced and daily up to sacrifice (approximately 1 hour after dosing on Days 1, 2, 3 and 5)

Body weight
The animals were weighed at allocation (Day 1) and on sacrifice (Day 6).

- TERMINAL PHASE
Euthanasia method and lymph nodes collection
The animals were killed on Day 6, approximately 24 hours after BrdU injection, by carbon dioxide narcosis. No necropsy was performed on the sacrificed animals. Shortly after sacrifice of each animal, the auricular lymph nodes were rapidly excised, pooled on individual basis and individually collected in a solution of 2 % BSA-PBS [2 % bovine serum albumine (BSA) in phosphate buffered saline, PBS]. Cell suspensions were prepared for the evaluation of proliferation at lymph node as detailed below.

- DETERMINATION OF CELLULAR PROLIFERATION
Preparation of single cell suspension
A single cell suspension of lymph node cells (LNC) was prepared from each animal by gentle mechanical disaggregation and passage through a 70 μm nylon mesh. The suspensions thus obtained were centrifuged and each supernatant resuspended in 20 mL of 2 % BSA-PBS.

Measurement of BrdU content in lymphocytes DNA
BrdU was measured by ELISA using a commercial kit, according to manufacturer instructions. Briefly, 100 μl of the LNC suspension were added to the wells of a flat-bottom 96-well microplate in triplicate. Two replicate microplates were prepared. After fixation and denaturation of the LNC, 100 μl of anti-BrdU antibody labelled with peroxidase were added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and 100 μl of the substrate solution were then added and allowed to produce chromogen. The reaction was finally stopped by adding 25 μl of stop solution (1 M H2SO4). Absorbance (OD) was detected at 450 nm (with reference wavelength: 690 nm). The replicate plate was destroyed after the evaluation of the results obtained in the first plate since the objective of the study had been achieved
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Differences between each treated group and the concurrent negative control group (individual BrdU labelling indices) were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test
Positive control results:
In the group treated with the positive control item, a Stimulation Index of 2.23 was calculated. As it was greater than 2, the study was regarded as valid
Parameter:
SI
Remarks on result:
other: The calculated Stimulation Indices were 1.29, 1.17 and 1.20 at low, mid- and high dose levels, respectively
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: No information available

INTERPRETATION OF DATA

Calculation

The BrdU labelling index is defined as follows:

BrdUlabelling index= (OD450ODblank450)(OD690ODblank690)

The BrdU labelling index was calculated for each mouse and a group mean was subsequently calculated. Results for each treatment group were expressed as the mean Stimulation Index (SI). The SI was derived by dividing the mean BrdU labelling index/mouse within each test item group and the positive control groups by the mean labelling indices for the respective vehicle group.

 

Criteria of interpretation for test item induced response

The test item is considered to induce sensitisation when the SI for any single treatment dose group is ≥ 1.6. It is not required that an increased response is observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e.in case of borderline results with 1.6 ≤ SI ≤ 1.9).

 

Assay validity criteria

The assay is considered satisfactory if the Stimulation Index (SI) of the positive control group is higher than 2.0.

 

RESULTS

Preliminary test

Five concentrations (50, 25, 10, 5 and 2.5 % w/w) of the test item were selected to be used in the preliminary test. The concentrations of 50 and 25 % w/w were found to be administrable with difficulties due to the nature of the obtained formulations.

No signs of toxicity (clinical signs or body weight losses) were observed at any of the tested concentrations.

The evaluation of visible reactions showed no erythema at any of the concentrations investigated (50, 25, 10, 5 and 2.5 % w/w).

The evaluation of ear thickness indicated that no increase was induced by treatment (values of Day 6 compared to Day 1). The evaluation of ear punch weight indicated that an increase, without any dose-relation, was observed in the animals treated at 2.5, 5, 25 and 50 % w/w ranging from 3 to 21 %, when compared to the animals treated with the vehicle. Based on the results described above and taking into consideration the difficulties to administer the highest concentration tested (50 % w/w), a 25 % w/w concentration was judged to be not irritant and selected for the main assay.

Main assay:-In vivophase

No mortality nor clinical signs were recorded in animals treated at all dose levels investigated (25, 10 and 5 % w/w). The administration of the concentration of 25 % w/w was found difficult.

Changes in body weight observed during the study were within the expected range for this strain and age of animals.

 

Main assay: Evaluation of cell proliferation

No significant increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated stimulation indices (SI) were 1.29, 1.17 and 1.20, at low, mid- and high dose levels respectively. In the group treated with the positive control item, a Stimulation Index of 2.23 was calculated. As it was greater than 2, the study was regarded as valid.

Interpretation of results:
not sensitising
Remarks:
Migrated information according to the CLP Regulation Criteria used for interpretation of results: EU
Conclusions:
Calculated Stimulation Index:
1.29, low dose level
1.17 mid dose level
1.20 high dose level
Executive summary:

Method

The potential of the test item, AP 1300 S, to cause skin sensitisation reactions following topical application to the skin of CBA/JN mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline for testing of chemicals No. 442b.

 

Observations

Five concentrations were tested in the preliminary test [50, 25, 10, 5 and 2.5 % w/w in acetone:olive oil 4:1 (v/v)] in order to identify a non toxic and minimally irritant concentration and avoid false positive results. No signs of toxicity (clinical signs or body weight losses) were observed at the tested concentrations. Concentrations of 50 % and 25 % w/w were found administrable with difficulty due to the nature of the formulations.

According to the results obtained and taking into consideration the difficulties to administer the highest concentration, a 25 % w/w concentration was judged to be not irritant and selected for the main assay.

In the main assay, the test item was topically administered at the concentrations of 25, 10 and 5% w/w in acetone:olive oil 4:1 (v/v).

No mortality nor clinical signs were recorded in any animal. Body weight changes were considered not remarkable. No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated stimulation indices (SI) were 1.29, 1.17 and 1.20 at low, mid- and high dose levels, respectively.

Results

The test item is considered as Not sensitising

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

The potential of the test item, AP 1300 S, to cause skin sensitisation reactions following topical application to the skin of CBA/JN mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline for testing of chemicals No. 442b.

No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices were 1.29, 1.17 and 1.20 at low, mid and high dose levels, respectively


Migrated from Short description of key information:
Not sensitising

Justification for selection of skin sensitisation endpoint:
Test conducted according to internationally accepted Guideline and in according to the GLP Principles

Justification for classification or non-classification

According to the OECD Guideline 442B, The test item is considered to induce sensitisation when the SI for any single treatment dose group is greate then 1.6. It is not required that an increased response is observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with SI between 1.6 and 1.9). Under the test condition the calculated stimulation indices (SI) were 1.29, 1.17 and 1.20 at low, mid and high dose levels, respectively, therefore the substance is Not classified as Skin Sensitizer.