2-(3-methoxypropyl)-9(or 10)-methylbenzimidazo[2,1-b]benzo[lmn][3,8]phenanthroline-1,3,6(2H)-trione

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: 02 November 2016 End of experimental phase: 14 November 2016 Study completion: 13 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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-(3-methoxypropyl)-9(or 10)-methylbenzimidazo[2,1-b]benzo[lmn][3,8]phenanthroline-1,3,6(2H)-trione

EC Number:

258-094-6

EC Name:

2-(3-methoxypropyl)-9(or 10)-methylbenzimidazo[2,1-b]benzo[lmn][3,8]phenanthroline-1,3,6(2H)-trione

Cas Number:

52673-15-1

Molecular formula:

C25H19N3O4

IUPAC Name:

17-(3-methoxypropyl)-6(or7)-methyl-3,10,17-triazahexacyclo[13.6.2.0²,¹.0,.0¹²,²².0¹,²³]tricosa-1(21),2,4(9),5,7,12(22),13,15(23),19-nonaene-11,16,18-trione

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
- 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: 9 week
- Weight at arrival: 18 to 22 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:02 November 2016 To:14 November 2016

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

TEST ITEM: 10, 5 and 2.5% (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 to evaluate the feasibility of test item formulations using acetone/olive oil 4:1 v/v, PEG and DMSO as
vehicle. A suspension suitable for application was obtained at a concentration of 10% w/w in acetone/olive oil 4:1 v/v.
- 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.

Results and discussion

Positive control results:

In the group treated with the positive control item, a Stimulation Index of 2.66 was calculated. As it was greater than 2, the study was regarded as valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.89

Variability:

0.023

Test group / Remarks:

2.5%

Results 2

Parameter:

SI

Value:

1.05

Variability:

0.024

Test group / Remarks:

5%

Results 3

Parameter:

SI

Value:

0.98

Variability:

0.023

Test group / Remarks:

10%

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 0.89, 1.05 and 0.98, respectively at low, mid- and high dose levels.

CLINICAL OBSERVATIONS: Neither mortality nor clinical signs were recorded in animals treated at all dose levels investigated [10, 5 and 2.5% (w/w)].

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

Applicant's summary and conclusion

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 0.89, 1.05 and 0.98, respectively at low, mid- and high dose levels.
In the group treated with the positive control item, a Stimulation Index of 2.66 was calculated. As it was greater than 2, the study was regarded as valid.

Executive summary:

The potential of the test item, Disperse Orange 032, to cause skin sensitisation reactions following topical application to the skin of CBA/JN mice, was assessed using the LLNA:BrdUELISA method, according to the OECD Guideline for testing of chemicals No. 442b.

Preliminary test

Five concentrations [10 (maximum feasible concentration), 5, 2.5, 1 and 0.5% 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 10% w/w was judged to be not irritant.

Main assay

In the main assay, the test item was topically administered at the concentrations of 10, 5 and 2.5% (w/w), in acetone:olive oil 4:1 (v/v).

No mortality nor clinical signswere recorded in any animal.

Changes in bodyweight 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 0.89, 1.05 and 0.98 at the low, midand high dose levels [2.5, 5 and 10 %,], 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