2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 July 2017 to 8 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

(due to technical reasons the actual temperature range was 19.1-25.6°C instead of 22 ± 3°C and the actual relative humidity range was 31-80% instead of 30-70%. These deviations 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:

2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

EC Number:

285-413-6

EC Name:

2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

Cas Number:

85098-62-0

Molecular formula:

C30H15ClN7Na5O15S4

IUPAC Name:

2-[[4-chloro-6-[[8-hydroxy-3,6-disulpho-7-[(1-sulpho-2-naphthyl)azo]-1-naphthyl]amino]-1,3,5-triazin-2-yl]amino]-5-sulphobenzoic acid, sodium salt

Test material form:

solid: particulate/powder

Remarks:

Dark brown powder

Details on test material:

- Storage conditions: Controlled room temperature (15 - 25 °C, below 70 RH%) in an opaque plastic flask.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old (age-matched, within one week)
- Weight at study initiation: 19.0 – 20.1 g
- Housing: Group caging in type II. polypropylene / polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days

In the Preliminary Experiment, mice of 8 weeks of age (18.3-19.3 g) were used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 - 25.6°C
- Humidity (%): 31 - 80%
- Air changes (per hr): 15 - 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Test material (formulated in 1% Pluronic) at 50, 25, and 10 % (w/v)

No. of animals per dose:

MAIN STUDY
4 animals per group (3 groups received test material at 50, 25, and 10 % (w/v). There was also a negative control group (vehicle only) and a positive control group)

Details on study design:

PRE-SCREEN TESTS:
2 animals/dose received test material at concentrations of 50 and 25 % (w/v) in 1% Pluronic. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.
Based on the observation of the solubility test, the maximum available concentration was 50 % (w/v).
In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
Based on the observations of the preliminary test, 50 % (w/v) dose was selected as top dose for the main test.

MAIN STUDY

- Topical application
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.

PROLIFERATION ASSAY
- Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and 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 (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
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. 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 mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

- Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating 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 resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

- Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated
resuspended and 3 mL of 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules.
After overnight (approximately 18 hours) incubation at 2 - 8°C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed. Pellets were resuspended in 1 mL of 5% (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid 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 duplicates by adding 1 mL of 5% (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

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 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.

EVALUATION OF THE RESULTS
DPM was measured for each pooled group of nodes. 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 “DPN” (DPM divided by the number of lymph nodes).
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

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

Since the test material gave a positive response and data permitted, the EC3 value of the test material was calculated (EC3 means the effective chemical concentration required for SI=3).

> Acceptability of the test
The Local Lymph Node Assay is considered valid if it meets the following criteria:
- the DPN value of the negative (vehicle) control group falls within the range of historical laboratory control data,
- the positive control substance produces a significant lymphoproliferative response increases (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)

Results and discussion

Positive control results:

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25% in the relevant vehicle (1% Pluronic) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 3.1) 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

Key result

Parameter:

SI

Value:

4.9

Test group / Remarks:

50% w/v

Results 2

Key result

Parameter:

SI

Value:

3.4

Test group / Remarks:

25% w/v

Results 3

Key result

Parameter:

SI

Value:

3.1

Test group / Remarks:

10% w/v

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The results of the proliferation assay are summarised in Table 1. The appearance of the lymph nodes was normal in the negative control group and in the 25 and 10% (w/v) test material treated dose groups. Larger than normal lymph nodes were observed in the 50% (w/v) test item treated group and in the positive control group.

CLINICAL OBSERVATIONS:
No mortality or signs of systemic toxicity were observed during the study. Alopecia around the ears was present in the 50% (w/v) dose group from Day 3 up to Day 6. Test material precipitate or minimal amount of test material precipitate was present in all test material-treated dose groups from Day 1 up to Day 6.

BODY WEIGHTS
No marked body weight losses (≥ 5%) was observed on the mean body weight changes in any groups.

INTERPRETATION OF OBSERVATIONS
The test material was powder, which was formulated in 1% Pluronic. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, 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 resulting stimulation indices observed under these exaggerated test conditions was considered to be good evidence that the test material is a sensitiser.

Any other information on results incl. tables

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

Test group	Measured DPM/ group	DPM	Number of lymph nodes	DPN	Stimulation index
Background (5 % (w/v) TCA)	32	-	-	-	-
Negative control (1% Pluronic)	1168	1136.0	8	142.0	1.0
Test material 50 % (w/v) in 1% Pluronic	5591	5559.0	8	694.9	4.9
Test mterial 25 % (w/v) in 1% Pluronic	3937	3905.0	8	488.1	3.4
Test mterial 10 % (w/v) in 1% Pluronic	3583	3551.0	8	443.9	3.1
Positive control (25 % (w/v) HCA in 1% Pluronic)	3558	3526.0	8	440.8	3.1

Applicant's summary and conclusion

Interpretation of results:

other: Skin Sens. 1B in accordance with EU criteria

Conclusions:

Under the conditions of the present assay the test material, tested in a suitable vehicle, was shown to have a sensitisation potential in the Local Lymph Node Assay. The extrapolated EC3 value of Reactive Red 31 is 7.4% (w/v). The test material requires classification for skin sensitisation (category 1B).

Executive summary:

The skin sensitiation potential of the test material was investigated in a GLP study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 429.

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 50 and 25% (w/v) in 1% Pluronic. Based on the observations recorded in the preliminary test, 50% (w/v) was selected as top dose for the main test.

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

- three groups received test material (formulated in 1% Pluronic) at 50, 25, and 10 % (w/v) concentrations respectively,

- the negative control group received the vehicle (1% Pluronic) only,

- the positive control group received 25 % (w/v) HCA (dissolved in 1% Pluronic).

The test material solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring disintegrations per minutes after incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

No mortality or signs of systemic toxicity were observed during the study. Alopecia around the ears was present in the 50% (w/v) dose group from Day 3 up to Day 6. Test material precipitate or minimal amount of test material precipitate was present in all treatment groups receiving test material formulations from Day 1 up to Day 6. No marked body weight losses (≥ 5%) was observed on the mean body weight changes in any groups.

The stimulation index values were 4.9, 3.4 and 3.1 at concentrations of 50, 25, and 10 % (w/v), respectively.

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historical positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

Under the conditions of the present assay the test material, tested in a suitable vehicle, was shown to have a sensitisation potential in the Local Lymph Node Assay. The extrapolated EC3 value of Reactive Red 31 is 7.4% (w/v).  The test material requires classification for skin sensitisation (category 1B).