Sodium 4-[4-[[2-methyl-4-[[(p-tolyl)sulphonyl]oxy]phenyl]azo]anilino]-3-nitrobenzenesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 06th to 12th, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 22th July 2010

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:

Acid Orange 067

IUPAC Name:

Acid Orange 067

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT, H-1103, Budapest, Cserkesz u. 90.
- Females: female, nulliparous, non-pregnant.
- Age at study initiation: 10-11 weeks old animals used in the preliminary test; 10-12 weeks old animals used in the main test.
- Weight at study initiation: 16.9 – 21.6 g. The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight.
- Housing during acclimatization period: grouped caging in small groups.
- Housing during the test: grouped caging (4 animals/cage).
- Cage type: type II. Polypropylene / polycarbonate.
- Bedding: laboratory bedding.
- Housing/Enrichment: mice were group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities.
- Diet: ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum.
- Water: tap water from watering bottles ad libitum.
- Acclimation period: 14 days.

ENVIRONMENTAL CONDITIONS
- Temperature:22 ± 3 °C
- Humidity: 30 – 70 %.
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

25, 10, 5 and 2.5 % (w/v)

No. of animals per dose:

PRELIMINARY STUDY: 6 animals (2 animals/groups)
MAIN TEST: 28 animals/main test (4 animals/treatment group)

Details on study design:

RANGE FINDING TESTS
The pre-experiments on formulation evaluation and the Dose Range Finding Test (DRF) were not performed in compliance with the GLP-Regulations.
The preliminary irritation/toxicity screen was conducted in a similar experimental manner to the exposure phase of the main test except there was no assessment of lymph node proliferation and fewer animals were used. The maximum test concentration was selected according to results of the preliminary formulation evaluation. The test item was examined in the DRF as 25 %, 10 % or 5 % (w/v) formulations (solutions) prepared with the selected vehicle of DMF.
No mortality, significant, treatment related effect on the body weights or any other sign of systemic toxicity were observed. No sign of significant irritatioN or any other local effect were observed.
Based on the preliminary test results the maximum attainable concentration (based on solubility) of 25 % (w/v) was used in the main test. The test item was tested also at three additional, lower concentrations (10 %, 5 % and 2.5 %, w/v) to evaluate dose-response relationship and ensure validity of the test in accordance with the relevant guidelines.

MAIN STUDY
In vivo treatment
Each mouse was topically treated with 25 µl of the appropriate formulations of the test item, the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear. After the treatments animals were returned to their cages. 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
No animals showed symptoms of systemic toxicity or excessive skin irritation, and no technical treatment failures were observed during the test: all animals treated were processed. Therefore no treatment group was excluded from the evaluation.

Injection of 3HTdR
On Day 6 each mouse was intravenously injected via the tail vein with 250 µl of sterile PBS (1 x PBS, diluted from 10x concentrate) containing approximately 20 µCi of
3H-methyl-thymidine using a hypodermic needle with 1 ml sterile syringe. Once injected, 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 sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps. Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish 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 lymph node cells (LNCs), pooled according to groups, 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). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 °C. After centrifugation, the supernatant was removed, leaving 1-2 ml supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final wash, each supernatant was removed leaving a small volume (< 0.5 ml) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 ml of 5 % (w/v) trichloroacetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8 °C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4 °C and decanting the supernatants, than the pellets were re-suspended in 1 ml of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 ml of scintillation liquid, gently mixed and loaded into the β-scintillation counter. 3HTdR incorporation was measured for up to 10 minutes per sample. The β-counter expressed the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 ml aliquots of 5 % TCA.

OBSERVATIONS
Clinical Observations
During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring during the whole test. Individual records were maintained.

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 (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value: the average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPM/mouse. The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Dose-response relationship was evaluated by linear regression using SI values. All calculations were made by Microsoft Excel Software. Based on the results an EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated.

INTERPRETATION OF RESULTS
The test item is considered as a skin sensitizer, if 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 (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

ASSAY ACCEPTANCE CRITERIA
- Lymph nodes from all 4 animals in each dose group were pooled for a valid experiment.
- Skin sensitizing effect was observed at the applied concentration of the positive control (the stimulation index was greater than 3).

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.5

Test group / Remarks:

at test item concentrations of 25 % (w/v)

Results 2

Parameter:

SI

Value:

0.9

Test group / Remarks:

at test item concentrations of 10 % (w/v)

Results 3

Parameter:

SI

Value:

0.9

Test group / Remarks:

at test item concentrations of 5 % (w/v)

Results 4

Parameter:

SI

Value:

2.1

Test group / Remarks:

at test item concentrations of 2.5 % (w/v)

Cellular proliferation data / Observations:

The maximum dose selection was based on results of the formulation evaluation and the Dose Range Finding test (DRF). Solubility of the test item in vehicles preferred in the LLNA was evaluated. Based on the results the test item was formulated in N,N-Dimethylformamide (DMF) in the LLNA. The maximum achievable concentration (based on solubility) was 25 % (w/v) using ultrasonic dispersion and stirring. According to results of the DRF (where no adverse effect was observed up to this maximum concentration) the test item was examined in the main test as 25 %, 10 %, 5 % or 2.5 % (w/v) formulations (apparently solutions) in DMF.
Since the test was valid and no confounding effects of irritation or systemic toxicity were observed during the main test, the proliferation values obtained are considered to reflect the real potential of the test item to cause/not cause lymphoproliferation in the Local Lymph Node Assay.
No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (DMF) was noted at the applied test concentrations. The observed stimulation index values were 0.5, 0.9, 0.9 and 2.1 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. No significant, biologically relevant dose-related response was observed.  

The assay acceptance criteria were fulfilled, hence the test was valid.

Proliferation assay
Since nofailed treatment, sign of systemic toxicity or irritation was observed during the test, thus no treatment group was excluded from the evaluation. Visually larger lymph nodes compared to the relevant vehicle control (AOO) were observed in the positive control group only. Appearance of the lymph nodes was normal in the negative control groups (AOO or DMF) and in the test item treated groups. No significant lymphoproliferative response (SI ≥ 3) compared to the relevant control (DMF) was noted for test item at the applied test concentrations. The observed stimulation index values were 0.5, 0.9, 0.9 and 2.1 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using the SI values.No statistical significance was observed (p = 0.26, r = 0.74).

Body Weight Measurement
No significant, treatment related effect on body weights was observed during the test. Body weights decreased by > 5 % were observed in the positive control group (1/4 animal, 7 % decrease) and in the 25 % (w/v) dose group (1/4 animals, 10 % decrease) but the effect was considered not significant since the mean body weights did not decrease significantly.

Clinical and Other Observations, Signs of Irritation
No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group.

Controls
The positive control group animals were treated with 25 % (w/v) HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. Significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 7.5). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay

Applicant's summary and conclusion

Interpretation of results:

other: not classified, according to the CLP Regulation (EC) No 1272/2008

Conclusions:

no skin sensitization

Executive summary:

The aim of this study was to evaluate the skin sensitization potential of test item following dermal exposure in the Local Lymph Node Assay. Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration. Solubility of the test item in vehicles preferred in the LLNA was evaluated.

Based on the results the test item was formulated in N,N-Dimethylformamide (DMF) in the LLNA. The maximum achievable concentration (based on solubility) was 25 % (w/v) using ultrasonic dispersion and stirring. According to results of the DRF (where no adverse effect was observed up to this maximum concentration) the test item was examined in the main test as 25 %, 10 %, 5 % or 2.5 % (w/v) formulations (apparently solutions) in DMF.

Appropriate positive control (α-Hexylcinnamaldehyde, HCA), furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.

The positive control item (25 % (w/v) HCA in Acetone:Olive oil 4:1 (v/v) mixture, AOO) induced significant stimulation over the relevant control (SI = 7.5) thus confirming the validity of the assay.

No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group.

No significant lymphoproliferation (SI ≥ 3) compared to the relevant control (DMF) was observed for the test item at the tested concentrations. The corresponding stimulation index values were 0.5, 0.9, 0.9 and 2.1 at test item concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. No significant, biologically relevant dose-response relationship was observed (p = 0.26, r = 0.74; evaluated by linear regression using the calculated SI values).

The lack of a significantly increased lymphoproliferation up to the maximum attainable concentration of 25 % (w/v, based on solubility) and also the lack of a significant, biologically relevant dose-response relationship are considered evidence that the substance is not a skin sensitizer.

Conclusion

Under the conditions of the assay, substance tested at the maximum feasible concentration of 25 % (w/v, based on solubility) and also at concentrations of 10 %, 5 % or 2.5 % (w/v) as formulations (apparently solutions) in a suitable vehicle (DMF) was shown to have no skin sensitization potential in the Local Lymph Node Assay.