9-[bis(2-hydroxyethyl)amino]-5-[(3-methylphenyl)amino]-7-phenylbenzo[a]phenazin-7-ium chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

8-[bis(2-hydroxyethyl)amino]-12-[(3-methylphenyl)amino]-10-phenyl-5,10λ⁵-diazatetraphen-10-ylium chloride

EC Number:

941-946-2

Molecular formula:

C33H31ClN4O2

IUPAC Name:

8-[bis(2-hydroxyethyl)amino]-12-[(3-methylphenyl)amino]-10-phenyl-5,10λ⁵-diazatetraphen-10-ylium chloride

Details on test material:

purity; 89.7 % (w/w) (main component)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS: mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 18.4 - 22.5 g
- Housing: group caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf, Germany), ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

Study design: in vivo (LLNA)

Vehicle:

other: ethanol/water (7+3, v/v)

Concentration:

5, 10, and 20% (w/w)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:

A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 20% suspension in ethanol/water (7+3, v/v). Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing).
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 20% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity. Redness of the ear skin could not be examined, due to the colour of the test item.
Thus, the test item in the main study was assayed at 5, 10, and 20% (w/w). The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

MAIN STUDY:

TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 5, 10, and 20% (w/w) in ethanol/water (7+3, v/v). The application volume, 25 µL was spread over the entire dorsal surface (∅ ~ 8 mm) of each ear once daily for three consecutive days. A futher group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF 3H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE

Five days after the first topical application, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (3HTdR). Approximately five hours after treatment with 3HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetíc acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a ß-scintillation counter.

INTERPRETATION OF RAW DATA

The proliferation response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiliser in the LLNA if the following criteria are fulfilled:
-First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
-Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

OBSERVATIONS

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
-Mortality / Viability: Once daily (week day) from experimental start to necropsy.
-Body weights: Prior to the first application and prior to sacrifice.
-Ear weights: After sacrifice biopsy punches were taken from each ear.
-Clinical signs (local / systemic): In the main experiment clinical signs were recorded within 1 hour after each application, and 24 ± 4 hours after the first and second application as well as on the day of preparation.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:

Experiment performed in October 2014 (Harlan study number 1486301) using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 1.32, 2.28, and 5.14, respectively.
The EC3 value calculated was 13.8 % (w/v).
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

In vivo (LLNA)

Results

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see depicted table below

Any other information on results incl. tables

Calculation and results of individual data; Vehicle: ethanol/water (7+3, v/v)

 

Test item concentration % (w/w)	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	9	---	---	---	---
---	BG II	14	---	---	---	---
0	1	6181	6169.5	8	771.2	1.00
5	2	5275	5263.5	8	657.9	0.85
10	3	4598	4586.5	8	573.3	0.74
20	4	3785	3773.5	8	471.7	0.61

1    =  Control Group

2-4=  Test Group

^a)   =  The mean value was taken from the figures BG I and BG II

^b)    =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period. Redness of the ear skin could not be examined, due to the colour of the test item. Black faeces was observed in all test item treated animals on day 2 and 3.

BODY WEIGHTS

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A statistically significant or biologically relevant increase in ear weights was not observed.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test itemwas not a skin sensitiser under the test conditions of this study.

Executive summary:

In order to study a possible skin sensitising potential of the test item, three groups each of four female mice were treated once daily with the test item at concentrations of 5, 10, and 20% (w/w) in ethanol/water (7+3, v/v) by topical application to the dorsum of each ear for three consecutive days. A control group of four mice was treated with the vehicle (ethanol/water (7+3, v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of³H-methyl thymidine measured in ß-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed. Redness of the ear skin could not be examined, due to the colour of the test item. Black faeces was observed in all test item treated animals on day 2 and 3. A statistically significant or biologically relevant increase in ear weights was not observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 0.85, 0.74, and 0.61 were determined with the test item at concentrations of 5, 10, and 20% (w/w) in ethanol/water (7+3, v/v). The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.