Sulfonation products of (N-phenyl-4-[[4-(phenylamino)phenyl][4-(phenylimino)cyclohexa-2,5-dien-1-ylidene]methyl]aniline, monohydrochloride and 4,4'-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]bis[N-phenylaniline], monohydrochloride), sodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 January 2017 - 24 January 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

pentasodium 4-({4-[(4-aminophenyl)({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene})methyl]phenyl}amino)benzene-1-sulfonate 4-{[4-({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene}({4-[(4-sulfonatophenyl)amino]phenyl})methyl)phenyl]amino}benzene-1-sulfonate

EC Number:

944-086-6

IUPAC Name:

pentasodium 4-({4-[(4-aminophenyl)({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene})methyl]phenyl}amino)benzene-1-sulfonate 4-{[4-({4-[(4-sulfonatophenyl)amino]cyclohexa-2,5-dien-1-ylidene}({4-[(4-sulfonatophenyl)amino]phenyl})methyl)phenyl]amino}benzene-1-sulfonate

Test material form:

solid

Details on test material:

UVCB

Specific details on test material used for the study:

Information as provided by the Sponsor.
Other name: Sulfonation product of (N-phenyl-4-[[4-phenylamino)phenyl]-[4-(phenylimino)cyclohexa-2,5-dien-1-ylidene]methyl]aniline, monohydrochloride and 4.4’-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]bis[N-phenylaniline], monohydrochloride), sodium salts
CAS No.: Not available
EINECS / EC No.: 944-086-6
Certificate of Analysis: AZ 909/Toxd3
Batch: DDIBLN519
Purity: Not indicated by the Sponsor
Physical state, appearance: Dark violet powder
Homogeneity: The test item appeared to be homogeneous
Expiry Date: 11 November 2024 (Statement of producer)
Storage Conditions:
(provided by the Sponsor) At room temperature
Stability in Solvent: Not indicated by the sponsor
Dose calculation not adjusted to purity.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 ± 2°C, relative humidity approx. 45-65%, artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

5, 10, and 20%

No. of animals per dose:

4

Details on study design:

Vehicle and Dose Selection
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 (3+7 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, sonicating, warming to 37°C).
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 (for detailed results see Appendix 1).
At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity.
Thus, the test item in the main study was assayed at 5, 10, and 20%. 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.

Test Item Preparation
The test item was placed into an appropriate container on a tared balance and ethanol/water (3+7 v/v) was added (w/w).
The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer.
The preparations were made freshly before each dosing occasion.

Test Item Administration
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 20% in ethanol/water (3+7 v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (  8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.8 µCi of 3H-methyl thymidine (equivalent to 79.2 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Terminal Procedure
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group).

Preparation of Single Cell Suspensions
Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.
Determination of cellular proliferation (incorporation of 3HTdR)
The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Observations
Clinical Observations
All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.

Determination of Ear Thickness
In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.

Determination of Ear Weights
In the pre-test and main experiment, after the lymph nodes have been excised, both ears of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance. The values obtained were taken down manually. The results are described in the report.

Determination of Body Weights
The body weights will be recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment)

Data Evaluation
Interpretation of raw data
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser 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.

Positive control substance(s):

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

Results and discussion

Positive control results:

October 2016 (study 1792800): S.I. of 1.50, 3.84, and 11.76 were derived at tested concentrations of 5, 10, 25%, respectively and an EC3 value of 8.2% was calculated.

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

>= 1.64 - <= 2.88

Test group / Remarks:

all

Remarks on result:

other: see depicted table below

Any other information on results incl. tables

Calculation and Results of Individual Data

Vehicle: ethanol/water (3+7 v/v)

Test item concentration %		Group		Measurement DPM		DPM-BG^a)		number of lymph nodes		DPM per lymph node^b)		S.I.
---		BG I		28		---		---		---		---
---		BG II		19		---		---		---		---
0		1		5886		5862.5		8		732.8		1.00
5		2		9615		9591.5		8		1198.9		1.64
10		3		16891		16867.5		8		2108.4		2.88
20		4		14163		14139.5		8		1767.4		2.41

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

 Calculation of the EC3 value

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.

  Body Weights

The body weight of the animals, recordedprior to the first application and prior to treatment with³HTdR, 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 relevant increase in ear weights was not observed.

Applicant's summary and conclusion

Conclusions:

The test item was 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% in ethanol/water (3+7 v/v) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could technically be achieved. A control group of four mice was treated with the vehicle (ethanol/water (3+7 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 ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed. A statistically significant increase in ear weight 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 1.64, 2.88, and 2.41 were determined with the test item at concentrations of 5, 10, and 20% in ethanol/water (3+7 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.