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Description of key information

No significant increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices (SI) were 1.27, 1.15 and 1.16 at low, mid and high dose levels, respectively. Hence the test item does not have skin sensitising properties.

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:
Start of experimental phase: 06 July 2016 End of experimental phase: 19 July 2016 Study completion: 12 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Version / remarks:
adopted on 22 July 2010
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Species:
mouse
Strain:
CBA:JN
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River France Laboratories, Domaine des Oncins B.P. 0109, F 69592 L’ARBRESLE CEDEX, France.
- Females (if applicable) nulliparous and non-pregnant: [yes/no/not specified] yes
- Age at study initiation: 8 week
- Weight at arrival: 17 to 20 grams
- Housing:Up to 5 during acclimatisation; 1/cage during the study
- Diet (e.g. ad libitum): Ad libitum throughout the study 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
- Water (e.g. ad libitum): Ad libitum
- Acclimation period:At least 5 days
- Indication of any skin lesions: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 °C±2 °C
- Humidity (%):55%±15%
- Air changes (per hr):Approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light):daily light/dark cycle of 12/12 hours
- IN-LIFE DATES: From:06 July 2016 To:19 July 2016
Vehicle:
dimethyl sulphoxide
Concentration:
TEST ITEM: 25, 10 and 5% (w/w).
Positive Control: 25%
No. of animals per dose:
4 animals per dose.
Details on study design:
PRE-SCREEN 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.
Since no suitable solution/suspension was obtained using this vehicle, PEG400 and dimethylsulfoxide were tested at the concentrations of 50 and 25% w/w.
Using dimethylsulfoxide at the concentration of 25 %, an administrable liquid suspension was obtained.
- Irritation: The treated sites of all animals were examined daily (once before first dosing, before dosing on Days 2 and 3 and daily thereafter).
Irritation to the skin was assigned a numerical value according to the Erythema scores reported below.
- Systemic toxicity: 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, on Days 1 to 3 or at approximately the same time on the remaining days)
- Ear thickness measurements: The ear thickness was measured by a suitable micrometer on Day 1 (before dosing), on Day 3 (before dosing) and on Day 6.
- Erythema scores: Erythema and eschar formation Value
No erythema 0
Very slight erythema 1
Well defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LOCAL LYMPH NODE ASSAY (LLNA: BrdU-ELISA method)
- Criteria used to consider a positive 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).

TREATMENT PREPARATION AND ADMINISTRATION: The animals were treated for three consecutive days (Days 1, 2, 3) with each vehicle, test or control
item formulations.
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.
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. If data were found to be inhomogeneous, aModified t test (Cochran and Cox)
was applied.
Positive control results:
In the group treated with the positive control item, a Stimulation Index of 2.39 was calculated. As it was greater than 2, the study was regarded as valid.
Parameter:
SI
Value:
1
Variability:
SD 0,074
Test group / Remarks:
negative control
Parameter:
SI
Value:
1.27
Variability:
SD 0,02
Test group / Remarks:
low: 5%
Parameter:
SI
Value:
1.15
Variability:
SD 0.022
Test group / Remarks:
mid: 10%
Key result
Parameter:
SI
Value:
1.16
Variability:
SD 0.044
Test group / Remarks:
high: 25%
Parameter:
SI
Value:
2.39
Variability:
SD 0.081
Test group / Remarks:
positive control
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: No significant increase in cell proliferation of draining lymph nodes was observed in any treatment group.

DETAILS ON STIMULATION INDEX CALCULATION: The calculated Stimulation Indices (SI) were 1.27, 1.15 and 1.16 at low, mid and high dose levels, respectively.

CLINICAL OBSERVATIONS: Neither mortality nor clinical signs were recorded in animals treated at all dose levels investigated [25, 10 and 5% (w/w)].

BODY WEIGHTS: Changes in body weight observed during the study were within the expected range for this strain and age of animals.
Interpretation of results:
GHS criteria not met
Conclusions:
No significant increase in cell proliferation of draining lymph nodes was observed in any treatment group.
The calculated Stimulation Indices (SI) were 1.27, 1.15 and 1.16 at low, mid and high dose levels, respectively.
In the group treated with the positive control item, a Stimulation Index of 2.39 was calculated.
As it was greater than 2, the study was regarded as valid.
Executive summary:

The potential of the test item, Acid Blue 324, 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. Preliminary test

Five concentrations [25 (maximum feasible concentration), 10, 5, 2.5 and 1% w/w in dimethylsulfoxide] were tested in the preliminary phase, in order to identify a non toxic and minimally irritant concentration and avoid false positive results.

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

According to the results of the irritation screening, the concentration of 25% w/w was judged to be not irritant.

Main assay

In the main assay, the test item was topically administered at the concentrations of 25, 10 and 5% (w/w), in dimethylsulfoxide.

No mortality nor clinical signs were recorded in any animal.

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

No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices (SI) were 1.27, 1.15 and 1.16 at the low, midand high dose levels [5, 10 and 25 %,], respectively. Neither correlation with the doses nor statistical significance was observed.

The above results indicate that the test item does not elicit any sensitisation response in mice following dermal exposure. European Directives concerning the classification, packaging and labelling of dangerous substances (Council Regulation (EC)No. 1272/2008 and subsequent revisions) would indicate the following:

Classification Not required

Signal word None indicated

Hazard statement None indicated

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

The potential of the test item, Acid Blue 324, 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.

Preliminary test: Five concentrations [25 (maximum feasible concentration), 10, 5, 2.5 and 1% w/w in dimethylsulfoxide] were tested in the preliminary phase, in order to identify a non toxic and minimally irritant concentration and avoid false positive results. No signs of toxicity (significant clinical signs or body weight losses) were observed at the tested concentrations. According to the results of the irritation screening, the concentration of 25% w/w was judged to be not irritant.

Main assay

In the main assay, the test item was topically administered at the concentrations of 25, 10 and 5% (w/w), in dimethylsulfoxide.

No mortality nor clinical signs were recorded in any animal.

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

No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices (SI) were 1.27, 1.15 and 1.16 at the low, mid and high dose levels [5, 10 and 25 %], respectively. Neither correlation with the doses nor statistical significance was observed.

The above results indicate that the test item does not elicit any sensitisation response in mice following dermal exposure.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

No significant increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices (SI) were 1.27, 1.15 and 1.16 at low, mid and high dose levels, respectively.

European Directives concerning the classification, packaging and labelling of dangerous substances (Council Regulation (EC)No. 1272/2008 and subsequent revisions) would indicate the following:

Classification        Not required

Signal word        None indicated

Hazard statement None indicated

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