N-phenyl-N'-[(phenylamino)sulfonyl]urea

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 February 2018 - 08 June 2018 (Draft Report)

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)

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:

1-phenyl-3-(phenylsulfamoyl)urea

EC Number:

813-358-5

Cas Number:

4886-26-4

Molecular formula:

C13H13N3O3S

IUPAC Name:

1-phenyl-3-(phenylsulfamoyl)urea

Test material form:

solid: particulate/powder

Details on test material:

- State of aggregation:
- Particle size distribution:
- Mass median aerodynamic diameter (MMAD):
- Geometric standard deviation (GSD):
- Shape of particles:
- Surface area of particles:
- Crystal structure:
- Coating:
- Surface properties:
- Density:
- Moisture content:
- Residual solvent:
- Activation:
- Stabilisation:
- Other:

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo
San Pietro al Natisone (UD)
Zona Industriale Azzida, 57,
33049 Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF, without further details given
- Age at study initiation: 9 weeks
- Weight at study initiation: 20.0 -23.1 g
- Housing: individual
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 13 days
- Indication of any skin lesions: none reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.2 - 26.2 °C
- Humidity (%): 23 - 86 %
- Air changes (per hr): 15-20 /h
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

other: Dioxane:olive oil mixture (4:1 (v/v))

Concentration:

5 %
10%
25 %

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) per vehicle with the concentrations of 25 % and 10% (w/v) in 4:1 (v/v) dioxane:olive oil), or 4:1 (v/v) diglyme:olive oil. 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. The maximum concentration of 25% was justified by trial formulation and Sponsor’s information.

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.

The comparison of the two vehicles showed that the 4:1 dioxane:olive oil (v/v) gave a better positive control result than the 4:1 (v/v) diglyme:olive oil. Also, in this study, the formulations with 4:1 (v/v) diglyme:olive oil gave higher value of ear thickness than 4:1 dioxane:olive oil (v/v).
Dioxane:olive oil mixture (4:1 (v/v)) was selected by the Sponsor for vehicle of the main test. Based on these results of the preliminary experiment, 25% (w/v) dose was selected as top dose for the main test

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
PROLIFERATION ASSAY with 3HTdR according to Guideline
- Criteria used to consider a positive response:
The test item 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.

TREATMENT PREPARATION AND ADMINISTRATION:
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 L of sterile PBS (phosphate buffered saline) containing approximately 20 Ci 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).

Positive control substance(s):

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

Statistics:

Mean was calculated over the 4 replicates per dose.

Results and discussion

Positive control results:

The result of the positive control substance -Hexylcinnamaldehyde (HCA) dissolved in the same vehicle (DOO, 4:1 dioxane:olive oil) was used to demonstrate the appropriate performance of the assay [1]. The positive control substance was examined at a concentration of 25 % in the relevant vehicle 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 (stimulation index value of 10.5) of HCA observed in the study was in line with historical control data of the positive control substance using other vehicles and also the results observed in the vehicle compatibility study

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.5

Test group / Remarks:

25% in 4:1 dioxane:olive oil (v/v)

Results 2

Key result

Parameter:

SI

Value:

2.2

Test group / Remarks:

10% in 4:1 dioxane:olive oil (v/v)

Results 3

Key result

Parameter:

SI

Value:

1.7

Test group / Remarks:

5% in 4:1 dioxane:olive oil (v/v)

Cellular proliferation data / Observations:

No mortality or signs of systemic toxicity were observed during the study.

Test item precipitate was observed in 4 out of 4 animals in the 25% (w/v) dose group on Days 1, 2 and 3; and in 1 out of 4 animals in the 10% (w/v) dose groups on Days 1 and 2. No test item precipitate was observed on the ears of any other animals.
Slight decrease was observed in individual body weight in all treatment and positive control groups. However, as marked body weight loss (>5%) was observed in one animal of the negative control group, this fact was considered to have no toxicological relevance.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, under the conditions of the present assay, N-phenyl-N'-[(phenylamino)sulfonyl]urea did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Executive summary:

The aim of this study was to determine the skin sensitisation potential and potency of N-phenyl-N'-[(phenylamino)sulfonyl]urea following dermal exposure in mice. The study was performed with vertebrate animals as no full regulatoryin vitroalternative is available. The minimum number of animals was used, in accordance to the regulatory guidelines for animal welfare.

 

The object of this study is to determine the skin sensitisation potential and potency of the test item following dermal exposure. The study has been performed with vertebrate animals as the applied regulatoryin vitro alternative tests* showed inconclusive/equivocal results. Therefore, an in vivo study has been run to provide reliable information about the skin sensitisation potential and potency of the test item for regulatory acceptance.

 

*IMPORTANT NOTE: With Commission Regulation (EU) 2016/1688 adopted on the 20th September 2016, the testing strategy for the assessment of the skin sensitization potential of a new substance now includes mandatory in vitro testing before any in vivo testing can be carried out, if the in vitro tests are technically feasible. This applies to all substances to be tested for EU REACH and CLP, but not for other regulatory purposes.

  

The recommended vehicles by OECD 429 were considered inappropriate by the Sponsor due to stability reasons. Therefore, based on literature and available stability information of the Sponsor, the compatibility of two alternative vehicles (4:1 (v/v) dioxane:olive oil and 4:1 (v/v) diglyme:olive oil) were tested in a previous vehicle compatibility study (Citoxlab study code 17/223-037EEV). Both of them were confirmed as acceptable vehicles in the LLNA by use of positive control testing. The 25 % (w/v) formulations were the highest feasible concentration for both vehicles which were suitable for the test. The formulations appeared to be opalescent solution, by visual examination.

 

As requested by the Sponsor, both vehicles were used in the Preliminary Irritation/Toxicity Test. The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses per vehicle (2 animals/dose): 25, 10 % (w/v) in 4:1 dioxane:olive oil (v/v) and 25, 10 % (w/v) in 4:1 diglyme:olive oil (v/v).

 

Based on the Preliminary Compatibility Test, on the observations recorded in the preliminary test, scientific advice from Citoxlab Hungary Ltd. and in agreement with the Sponsor, 25% (w/v)in 4:1 dioxane:olive oil (v/v) mixture (abbreviated as DOO)was selected as top dose for the main test.

 

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

-   three groups received N-phenyl-N'-[(phenylamino)sulfonyl]urea (formulated inDOO)at 25, 10 and 5 % (w/v) concentrations respectively,

-   the negative control group received the vehicle (DOO) only,

-   the positive control group received 25 % (w/v)a-Hexylcinnamaldehyde (HCA, dissolved inDOO).

The test item 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. Test item precipitate was observed in 4 out of 4 animals in the 25 % (w/v) dose group on Days 1, 2 and 3; and 1 out of 4 animals in the 10 % (w/v) dose group on Days 1 and 2. No test item precipitation was observed on the ears of any other animals. No test item related effect was observed on body weight.

 

The observed stimulation index values were 1.5, 2.2 and 1.7 at concentrations of 25, 10 and 5 % (w/v), respectively.

 

The result of the positive control substance a-Hexylcinnamaldehyde dissolved in the same vehicle (4:1 (v/v) dioxane:olive oil) was used to demonstrate the appropriate performance of the assay and to justify the use of this non-standard vehicle. The lymphoproliferative response was in line with historical positive control data for the positive control chemical, this result confirmed the validity of the assay.