Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino(3-methylpropane-1,3-diyl)]]bis(benzenesulphonate)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 August 2015 to 01 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

- see below

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

- see below

Principles of method if other than guideline:

The temperature (17.4 to 26.8 °C) and relative humidity values (24 to 87 %) were outside the expected ranges during the study due to technical reasons. However, these differences in the environmental parameters were considered not to adversely affect the results or integrity of the study.

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:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino(3-methylpropane-1,3-diyl)]]bis(benzenesulphonate)

EC Number:

274-999-9

EC Name:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino(3-methylpropane-1,3-diyl)]]bis(benzenesulphonate)

Cas Number:

70900-27-5

Molecular formula:

C34H32N2O8S2.2Na

IUPAC Name:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino(3-methylpropane-1,3-diyl)]]bis(benzenesulphonate)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Appearance: Blue powder
- Storage conditions of test material: Controlled room temperature (15 to 25 °C, below 70 RH %), protected from light and humidity

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: CBA/CaOlaHsd
- Age at study initiation: 10 weeks old (age-matched, within one week)
- Weight at study initiation: 21.0 to 23.1 g (the weight variation in animals in the study did not exceed ± 20 % of the mean weight)
- Housing: Group caging in Type II polypropylene / polycarbonate cages; mice were provided with glass tunnel-tubes, bedding and nest building material
- Diet: ad libitum
- Water: Animals received tap water from the municipal supply from 500 mL bottles, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 17.4 to 26.8 °C
- Humidity: 24 to 87 %
- Air changes: 15 to 20 air exchanges/hour
- Photoperiod: 12 hours of light daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

other: 1 % aqueous Pluronic® PE9200 solution (1 % Pluronic)

Concentration:

25, 10 and 5 % (w/v) concentrations

No. of animals per dose:

4 females per dose

Details on study design:

PRELIMINARY SCREENING TEST
- Compound solubility
The solubility of the test material was examined and the following standard OECD vehicles were assessed: Acetone:Olive oil 4:1 (v:v) mixture (AOO), N,N-Dimethylformamide (DMF), Methyl ethyl ketone (MEK), Propylene glycol (PG), Dimethyl sulfoxide (DMSO) and 1 % aqueous Pluronic® PE9200 (1 % Pluronic). The 100 and 50 % (w/v) formulations were not achievable using any of these vehicles. The 25 % (w/v) formulation was achievable using 1 % Pluronic as vehicle, while the use of other vehicles did not result a suitable formulation at this concentration. Therefore, 1 % Pluronic was selected as vehicle for the study.

-Preliminary Irritation/Toxicity Test:
Performed using two doses (2 animals/dose), at test material concentrations of 25 and 10 % (w/v) in 1 % Pluronic. The preliminary experiment was conducted in a similar experimental manner to the main study, but they were terminated on Day 6 with a body weight measurement and the radioactive proliferation assay was not performed.
No mortality or signs of systemic toxicity were observed. No marked body weight loss (>5 %) was detected for any experimental animal. Test material precipitate was detected on the ears of the animals in both dose groups on Days 1 to 3.
Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination on Day 6. Increased ear thickness values were detected in some cases on Days 3 and/or Day 6 but the mean values were below the irritant response. The ear punch weights were within the historical control range. There was no indication of irritation at the site of application based on the visual assessment (erythema scoring).
The draining auricular lymph nodes of the animals were visually examined and they were found to be normal in both dose groups (subjective judgement by analogy with observations of former experiments).
Based on these results, 25 % (w/v) dose group was selected as the top dose for the main test.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION
-Test Material Preparation
Formulations were prepared on weight: volume basis as % (w/v) and were considered to be stable for this short period; no correction for purity of the test material was applied. To prepare the dosing formulations, the test material was freshly diluted with 1% Pluronic. Formulations were checked for visible homogeneity and physical stability.
-Test Material Administration
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

OBSERVATIONS
- Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed at least twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6.
- Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to ³HTdR injection) with a precision of ± 0.1 g.

PROLIFERATION ASSAY
- Injection of Tritiated Thymidine (³HTdR)
On Day 6 each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of ³HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

- Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanised by asphyxiation with ascending doses of carbon dioxide. The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes (deep anaesthesia was confirmed before making incision). The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of each mouse were collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. The nodes of each animal were processed individually.

- Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregation of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C.
After centrifugation supernatants were discarded. Pellets were gently re-suspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for the lymph nodes of each individual animal.

- Determination of Incorporated ³HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated, re-suspended and 3 mL of 5 % (w/v) trichloroacetic acid (TCA) solution was added to the tubes for precipitation of macromolecules. After overnight incubation at 2 to 8 °C (approximately 18 hours), precipitates were centrifuged (approximately 190 x g for 10 minutes at 4 °C), and supernatants were removed. Pellets were re-suspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a scintillation vial filled with 10 mL of scintillation liquid (Optiphase HiSafe 3) and thoroughly mixed. The vials were loaded into a β-scintillation counter and ³HTdR incorporation was measured (10-minute measurement).
The β-counter expresses the ³HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicate by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

EVALUATION AND INTERPRETATION OF THE RESULTS
The proliferative response of lymph node cells from the lymph nodes of each individual animal is expressed as radioactive disintegrations per minute (DPM) per animal.
A stimulation index of 3 or greater is an indication of a positive result.

- The test material is regarded as a sensitiser if both of the following criteria are fulfilled:
1. That exposure to at least one concentration of the test material resulted in an incorporation of ³HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
2. 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.

VALIDITY CRITERIA
- The DPN value of the negative (vehicle) control group falls within the range of historical laboratory control data
- The positive control produces a significant lymphoproliferative response increase (SI>3)
- Each treated and control group includes at least 4 animals
- The test material does not cause serious systemic or local toxicity.

Positive control substance(s):

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

Statistics:

The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value.
The results were expressed as disintegrations per node (DPN = DPM divided by the number of lymph nodes) for each animal following the industry standard for data presentation.
Stimulation index (SI = mean DPN of treated group divided by mean DPN of the appropriate control group) for each treatment group was also calculated.
The use of the individual approach to calculate the SI made the use of a statistical analysis available. The statistical analysis was performed using the SPSS/PC+ (4.0.1) software package. The heterogeneity of variance between groups was checked by Bartlett's test for the measured DPM values.
Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the result was positive, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by the Kolmogorow-Smirnow test. In the case of not normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using the Mann-Whitney U-test.

Results and discussion

Positive control results:

The positive control material was examined at a concentration of 25 % (w/v) in 1 % Pluronic. No mortality, cutaneous reactions or signs of toxicity were observed for the positive control material in the study. A significant lymphoproliferative response (stimulation index value of 6.9) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The group SI values were 1.0, 0.8, 0.6 and 0.8 for the control, 5, 10 and 25 % test material concentrations, respectively. The value for the positive control was 6.9.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The group DPN (DPN = DPM divided by the number of lymph nodes) values were 306.8, 231.8, 197.9 and 252.0 for the control, 5, 10 and 25 % test material concentrations, respectively. The value for the positive control was 2130.6.

Any other information on results incl. tables

Since there were no confounding effects of irritation or significant systemic toxicity at any examined concentration, the proliferation values obtained are considered to reflect the real potential of the test material to cause lymphoproliferation in the Local Lymph Node Assay.

The resulted mean stimulation index values observed under the test conditions were well below the threshold limit of 3, indicating that the test material is a not a skin sensitiser. The size of lymph nodes was in good correlation with this conclusion.

The DPN values observed for the negative (vehicle) control group and positive control group in this experiment were within the historical control range and the study was considered to be valid.

Table 1: DPM, DPN and Stimulation Index Values for all Groups

Test Group	Animal Number	Measured Total DPM	DPM	Number of lymph nodes	DPN	Group DPN	Mean Stimulation Index
Background (5 % (w/v) TCA)	-	36 38	-	-	-	-	-
Negative control (1 % Pluronic)	1 2 3 4	593 1237 411 361	556.0 1200.0 374.0 324.0	2 2 2 2	278.0 600.0 187.0 162.0	306.8	1.0
Test material 25 % (w/v) in 1 % Pluronic	5 6 7 8	575 550 477 562	538.0 513.0 440.0 525.0	2 2 2 2	269.0 256.5 220.0 262.5	252.0	0.8
Test material 10 % (w/v) in 1 % Pluronic	9 10 11 12	477 471 421 362	440.0 434.0 384.0 325.0	2 2 2 2	220.0 217.0 192.0 162.5	197.9	0.6
Test material 5 % (w/v) in 1 % Pluronic	13 14 15 16	672 835 190 305	635.0 798.0 153.0 268.0	2 2 2 2	317.5 399.0 76.5 134.0	231.8	0.8
Positive control (25 % HCA in 1 % Pluronic)	17 18 19 20	4949 2789 5282 4173	4912.0 2752.0 5245.0 4136.0	2 2 2 2	2456.0 1376.0 2622.5 2068.0	2130.6	6.9*

*Significant (p<0.05, Mann-Whitney U-test versus negative control)

 

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test material was considered to be a non-sensitiser under the conditions of this study.

Executive summary:

The potential of the test material to act as a sensitiser was investigated in a LLNA test conducted in accordance with the standardised guidelines OECD 429 and EU Method B.42 under GLP conditions.

The study assessed the skin sensitisation potential of the test material in female mice of the CBA/CaOlaHsd strain following topical application to the dorsal surface of the ear. Three groups, each of four animals, were treated on three consecutive days with 25 µL per ear of the test material formulated in 1 % aqueous Pluronic® PE9200 (1 % Pluronic) solution at concentrations of 25, 10 and 5 % w/w. A negative control group of four animals was treated with 1 % Pluronic alone. A concurrent positive control test, using a group of four animals, was performed with 25 % (w/v) HCA (dissolved in 1% Pluronic).

The group DPN (DPN = DPM divided by the number of lymph nodes) values were 306.8, 231.8, 197.9 and 252.0 for the control, 5, 10 and 25 % test material concentrations, respectively. The value for the positive control was 2130.6.

The group SI values were 1.0, 0.8, 0.6 and 0.8 for the control, 5, 10 and 25 % test material concentrations, respectively. The value for the positive control was 6.9.

Since there were no confounding effects of irritation or significant systemic toxicity at any examined concentration, the proliferation values obtained are considered to reflect the real potential of the test material to cause lymphoproliferation. The resulting mean stimulation index values observed were well below the threshold limit of 3, indicating that the test material is a not a skin sensitiser. The size of lymph nodes was in good correlation with this conclusion.

The DPN values observed for the negative (vehicle) control group and positive control group in this experiment were within the historical control range and the study was considered to be valid.

Under the conditions of this study, the test material was considered to be a non-sensitiser.