4',4'''-azobis[N-(9,10-dihydro-9,10-dioxo-1-anthryl)[1,1'-biphenyl]-4-carboxamide]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of experimental phase: 28 June 2017 End of experimental phase: 10 July 2017 Study completion: 02 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

4',4'''-azobis[N-(9,10-dihydro-9,10-dioxo-1-anthryl)[1,1'-biphenyl]-4-carboxamide]

EC Number:

235-436-2

EC Name:

4',4'''-azobis[N-(9,10-dihydro-9,10-dioxo-1-anthryl)[1,1'-biphenyl]-4-carboxamide]

Cas Number:

12227-50-8

Molecular formula:

C54H32N4O6

IUPAC Name:

4',4'''-azobis[N-(9,10-dihydro-9,10-dioxo-1-anthryl)[1,1'-biphenyl]-4-carboxamide]

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:JN

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Species and strain: Mice, CBA/JN
Sex: Females (nulliparous and non-pregnant)
Age: 7 weeks old
Supplier: Charles River Italia S.p.A., Calco (Lecco), Italy
Breeder: Charles River France Laboratories, Domaine des Oncins B.P. 0109, F 69592 L’ARBRESLE CEDEX, France.
Date of arrival: 15 June 2017
Weight range at arrival: 20.1 to 22.2 grams (differently from that indicated in the Study Protocol)
Acclimatisation period: At least 5 days
Veterinary health check: During acclimatisation period

ENVIRONMENTAL CONDITIONS
Animals per cage: Up to 5 during acclimatisation; 1/cage during the study
Housing: Polysulfone solid bottomed cages measuring 35.5 × 23.5 × 19 cm with nesting material
Cage control: Daily inspected and changed as necessary (at least twice/week)
Water: drinking water supplied to each cage via a water bottle
Water supply: ad libitum
Diet: 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
Diet supply: ad libitum throughout the study
Room lighting Artificial: (fluorescent tubes), daily light/dark cycle of 12/12 hours
Air changes: Approximately 15 to 20 air changes per hour
Temperature range: 22 °C±2 °C
Relative humidity range: 55%±15%

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Test item: 25, 10 and 5% (w/w).
Positive control: 25% (w/w)

No. of animals per dose:

4 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. At concentration of 25% w/w in acetone/olive oil 4:1 v/v, an administrable homogeneous emulsion 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).
- 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, when applicable)
- 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:Irritation to the skin was assigned a numerical value according to the table below:
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 the vehicle or test item formulations. 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.

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, a Modified t test (Cochran and Cox) was applied.

Results and discussion

Positive control results:

In the group treated with the positive control item, a Stimulation Index of 11.84 was calculated. As it was greater than 2, the study was regarded as valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

3.52

Test group / Remarks:

5%

Results 2

Parameter:

SI

Value:

4.28

Test group / Remarks:

10%

Results 3

Parameter:

SI

Value:

3.96

Test group / Remarks:

25%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: An increase in cell proliferation of draining lymph nodes was observed in the low, mid- and high dose groups. This increase reached statistical significance at all dose levels (5, 10 and 25% w/w).

DETAILS ON STIMULATION INDEX CALCULATION: The calculated Stimulation Indices (SI) were 3.52, 4.28 and 3.96, at the low, mid and high dose levels [5, 10 and 25%], respectively.

CLINICAL OBSERVATIONS: One positive control animal was found dead in cage on Day 5 of study at 1 hour after dosing with the 5-Bromo-2’-Deoxyuridine. The cause of death was probably due to a misdosing.
No clinical signs were recorded in animals treated at all dose levels investigated [25, 10 and 5% (w/w)].
However, the high SI values observed in the 3 surviving animals demonstrated the validity of the test.

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

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The potential of the test item, Vat Yellow 33, 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.
An increase in cell proliferation of draining lymph nodes was observed in the low, mid- and high dose groups. The calculated Stimulation Indices were 3.52, 4.28 and 3.96, at the 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 Category 1
Signal word Warning
Hazard statement H317: May cause an allergic skin reaction

Executive summary:

The potential of the test item, Vat Yellow 33, 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 acetone:olive oil 4:1 (v/v)] 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 acetone:olive oil 4:1 (v/v). One animal of the positive control group was found dead in cage.

No 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.

An increase in cell proliferation of draining lymph nodes was observed in the low, mid- and high dose groups. This increase reached statistical significance at all dose levels (5, 10 and 25% w/w). The calculated Stimulation Indices (SI) were 3.52, 4.28 and 3.96, at the low, midand high dose levels [5, 10 and 25%], respectively.

The above results indicate that the test item may elicit a sensitisation response in mice following dermal exposure, since in all dose groups the Stimulation Index was greater than 1.6. 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 Category 1

Signal word Warning

Hazard statement H317: May cause an allergic skin reaction