3-[(phenylcarbamoyl)amino]phenyl 4-methylbenzenesulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 April 2017 - 30 May 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)

Version / remarks:

22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

6 July 2012

Deviations:

no

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:

3-(3-phenylureido)phenyl 4-methylbenzenesulfonate

Cas Number:

2102522-55-2

Molecular formula:

C20H18N2O4S

IUPAC Name:

3-(3-phenylureido)phenyl 4-methylbenzenesulfonate

Test material form:

solid: particulate/powder

Details on test material:

Appearance: Off-white powder
Storage conditions: Controlled room temperature (15-25 °C, below 70 RH %)

Specific details on test material used for the study:

No correction for purity of the test material was applied.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

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: 20.5 - 22.0 g
- Housing: Group caging in Type II polypropylene/polycarbonate cages. Mice were additionally provided with glass tunnel-tubes.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days
- Indication of any skin lesions: Not specified, only healthy animals were used for the study

In the Preliminary Experiment, mice of 10 weeks of age (21.3-22.9 g) were used.

ENVIRONMENTAL CONDITIONS
- Temperature: 18.2 – 25.9 °C
- Humidity: 25 - 80 %
- Air changes: 15 -20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

- Test material: 10, 25, 50 % (w/v)

No. of animals per dose:

Test material: 4
Negative (vehicle) control (DMSO):4
Positive control (25 % α-Hexylcinnamaldehyde (HCA) in DMSO): 4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test material was examined in a short Preliminary Compatibility Test. The following standard OECD vehicles were assessed: acetone: olive oil 4:1 (v/v) mixture, N,Ndimethylformamide, methyl ethyl ketone, propylene glycol, dimethyl sulfoxide (DMSO) and 1 % aqueous Pluronic® PE9200. The best vehicle taking into account the test material characteristics, its usage and the requirements of the relevant OECD guideline was considered to be DMSO. The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test material was 50 % (w/v). The formulations appeared to be clear solutions by visual examination. The test material was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)) in the test facility.

- Preliminary Irritation/Toxicity Test
- The Preliminary Irritation/Toxicity Test was conducted on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test material concentrations of 50 and 25 % (w/v) in DMSO. 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.
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 and scored. 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.
No mortality or signs of systemic toxicity were observed. A visible amount of test material precipitation was observed on the ear of the animals (2 out of 2, respectively) from Day 1 up to Day 6 in the 50 % (w/v) group, and from Day 1 to Day 4 in the 25 % (w/v) group. Rigid ears or slightly rigid ears were observed from Day 2 up to Day 6 in the 50 % (w/v) group, and Day 2 (1 out of 2 animals) and Day 3 (2 out of 2 animals) in the 25 % (w/v) group. There were no indications of any irritancy at the site of application.
Marked body weight loss (>5% reduction of body weight) was observed in one animal from each group, bringing the average body weight loss over 5 % in the 25 % (w/v) group.
The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups (subjective judgement by analogy with observations of former experiments).
Ear thickness measurements: The ear thickness values on Day 6 were significantly (more than 25 % compared to the Day 1 values) larger in the 50 % (w/v) dose group (3 out of 4 measurements). Ear punch weights were within the acceptable range.
Erythema scores: 0 on Days 1 - 6.
Based on these observations, 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 euthanized 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. 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 4°C), 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. Individual records were maintained.
- 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) following the industry standard for data presentation.
- 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: The test material is regarded as a sensitizer 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.

- Acceptability of the test: The Local Lymph Node Assay is considered valid if it meets the following criteria:
1) the DPN value of the negative (vehicle) control group falls within the range of historical laboratory control data,
2) the positive control substance produces a significant lymphoproliferative response increases (SI > 3),
3) each treated and control group includes at least 4 animals,
4) 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:

- 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 5.8) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
- Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were in within the historical control range. Each treated and control group included 4 animals.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.6

Test group / Remarks:

50 % (w/v) test material in DMSO

Results 2

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

25 % (w/v) test material in DMSO

Results 3

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

10 % (w/v) test material in DMSO

Cellular proliferation data / Observations:

CLINICAL OBSERVATION
- No mortality or signs of systemic toxicity were observed during the study. Rigid ears or slightly rigid ears from Day 3 up to Day 5 (4 out of 4 animals), alopecia around the ears from Day 4 up to Day 6 (3 out of 4 animals) were observed in the 50 % (w/v) dose group. Rigid ears or slightly rigid ears were observed on Day 3 in the 25 % (w/v) dose group.
- A visible amount of test material precipitation was observed on the ear of the test material treated animals from Day 1 up to Day 6 (15 out of 15 animals).

BODY WEIGHT MEASUREMENT
- No marked body weight losses (≥5 %) was observed on the mean body weight changes; however, the body weight loss was ≥5 % for 1/4 and 2/4 animals in the 50 % (w/v) and in the positive control groups, respectively. These changes were considered incidental.

PROLIFERATION ASSAY
- The appearance of the lymph nodes was normal in the negative control group and in the 50, 25 and 10 % (w/v) test material treated dose groups. Larger than normal lymph nodes were observed in the positive control group.

INTERPRETATION OF OBSERVATIONS
- The test material was powder, which was formulated in DMSO. 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 non-sensitizer. The size of lymph nodes was in good correlation with this conclusion.
- Based on the observed results, the test material does not need classification according to the GHS or CLP.

RELIABILITY OF THE TEST
- 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 (DMSO) 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 5.8) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
- Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were in within the historical control range. Each treated and control group included 4 animals.

Any other information on results incl. tables

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Measured DPM / group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5 % (w/v) TCA)	30.5	-	-	-	-
Negative control (DMSO)	3555	3524.5	8	440.6	1.0
Test Material 50 % (w/v) in DMSO	9243	9212.5	8	1151.6	2.6
Test Material 25 % (w/v) in DMSO	4383	4352.5	8	544.1	1.2
Test Material 10 % (w/v) in DMSO	3044	3013.5	8	376.7	0.9
Positive control (25 % (w/v) HCA in DMSO)	20487	20456.5	8	2557.1	5.8

Applicant's summary and conclusion

Interpretation of results:

other: not classified according to EU criteria

Conclusions:

Under the conditions of the test material did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 429 and EU Method B.42, under GLP conditions.

A Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 50 and 25 % (w/v) in DMSO. Based on the observations recorded in the preliminary test, the 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 the test material (formulated in DMSO) at 50, 25 and 10 % (w/v) concentrations respectively,
- the negative control group received the vehicle (DMSO) only,
- the positive control group received 25 % (w/v) HCA (dissolved in DMSO).
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. No marked body weight loss (≥5%) was observed on the mean body weight changes, although 1/4 and 2/4 animals showed marked body weight loss in the 50 % (w/v) and positive control groups, respectively. These changes were considered incidental.

The stimulation index values for the test material were 2.6, 1.2 and 0.9 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.

Overall, the test material did not show a sensitisation potential (non-sensitizer) in this Local Lymph Node Assay.