2,2'-(2,5-dichloro-1,4-phenylene)bis(1H-anthra[1,2-d]imidazole-6,11-dione)

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: 13 September 2017 End of experimental phase: 25 September 2017 Study completion: 20 November 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 (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-(2,5-dichloro-1,4-phenylene)bis(1H-anthra[1,2-d]imidazole-6,11-dione)

EC Number:

255-078-0

EC Name:

2,2'-(2,5-dichloro-1,4-phenylene)bis(1H-anthra[1,2-d]imidazole-6,11-dione)

Cas Number:

40783-05-9

Molecular formula:

C36H16Cl2N4O4

IUPAC Name:

2-(2,5-dichloro-4-{6,11-dioxo-1H,6H,11H-anthra[1,2-d]imidazol-2-yl}phenyl)-1H,6H,11H-anthra[1,2-d]imidazole-6,11-dione

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, Jackson, USA.
Date of arrival: 07 September 2017
Weight range at arrival: 17 to 20 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: 50, 25 and 10% (w/w).
Positive control: approximately 29% (w/w)

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. At the concentration of 50%w/w in acetone:olive oil 4:1 v/v, a homogeneous creamy formulation 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:
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 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 or a syringe.

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.

Results and discussion

Positive control results:

In the group treated with the positive control item, a Stimulation Index of 4.66 was calculated. As it was greater than 2, the study was regarded as valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.95

Test group / Remarks:

10% (w/w)

Results 2

Parameter:

SI

Value:

1.26

Test group / Remarks:

25% (w/w)

Results 3

Parameter:

SI

Value:

1.08

Test group / Remarks:

50% (w/w)

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 0.95, 1.26 and 1.08, respectively at low, mid- and high dose levels.

CLINICAL OBSERVATIONS: One mid-dose animal was found dead on Day 6 of study. The cause of death was probably due to a misdosing with the 5-Bromo-2’-Deoxyuridine. No clinical signs were recorded in animals treated at all dose levels investigated [50, 25 and 10% (w/w)].

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:

GHS criteria not met

Conclusions:

The potential of the test item, Vat Yellow 46, 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 0.95, 1.26 and 1.08 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

Executive summary:

The potential of the test item, Vat Yellow 46, 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 [50 (maximum feasible concentration), 25, 10, 5 and 2.5% 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 50% w/w was judged to be not irritant.

Main assay

In the main assay, the test item was topically administered at the concentrations of 50, 25 and 10% (w/w), in acetone:olive oil 4:1 (v/v). One animal of the mid-dose group was found dead on Day 6.

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.

No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated Stimulation Indices (SI) were 0.95, 1.26 and 1.08 at the low, mid- and high dose levels [10, 25 and 50 %,], 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