3-methyl-1,1-diphenylurea

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11.02.-24.02.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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-methyl-1,1-diphenylurea

EC Number:

236-039-7

EC Name:

3-methyl-1,1-diphenylurea

Cas Number:

13114-72-2

Molecular formula:

C14H14N2O

IUPAC Name:

3-methyl-1,1-diphenylurea

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material: 3-Methyl-1,1-diphenylurea- Physical state: white solid powder- Composition of test material, percentage of components: main component: 3-Methyl-1,1-diphenylurea CAS:13114-72-2 >98% (w/w) impurities: unlisted additives: unlisted-Molecular formula: C14H14N2O-Molecular weight: 226,27-Batch No.: 14015- Expiration date of the lot/batch: 11/2019- Stability under test conditions: stable- Storage condition of test material: in closed container, in dry room at room temperature (at laboratory conditions)

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS- Source: Breeding farm VELAZ s.r.o., Unětice 139, 252 62, Czech Republic, RČH CZ 21760118- Age at study initiation: 8 to 10 weeks (at start of dosing)- Weight at study initiation: 18.27 – 20.18 g (at start of dosing), in pilot experiment 17.64 – 18.22 g- Housing: Animals in groups: maximum six in macrolon cages (35x20x15 cm) with sterilized softwood shavings- Diet: ad libitum, drinking tap water- Water: ad libitum, Pelleted standard diet for experimental animals ad libitum (ST - 1, VELAS a.s., Hrabanov 535, 289 22 Lysá nad Labem, CZ 801080-01). Microbiological control and content of nutrients is performed - Acclimation period: at least 15 daysENVIRONMENTAL CONDITIONS- Temperature (°C): Room temperature: 22 ± 3 °C, permanently monitored - Humidity (%): Relative humidity: 30 – 70 %, permanently monitored - Air changes (per hr): 15 air changes per hours- Photoperiod (hrs dark / hrs light): Light: 12 hours light/dark cycle: 6am-6pm/6pm-6amSTUDY TIME SCHEDULEPilot experiment:Animal arrival/start of acclimatization: 28. 01. 2015Pilot experiment: 11. 02. – 16. 02. 2015Main study:First day of administration: 18. 02. 2015End of treatment period: 20. 02. 2015Application of radionuclide and necropsy: 23. 02. 2015Experimental completion date: 24. 02. 2015Final report completion: 10. 03. 2015

Study design: in vivo (LLNA)

Vehicle:

other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol

Concentration:

50% (w/v)500 mg/mL 5% (w/v)50 mg/mL0.5% (w/v)5 mg/mL

No. of animals per dose:

5 animals

Details on study design:

PILOT EXPERIMENTThe test item was administered to three animals to assess a possible systemic toxicity or high irritation to skin. The test item was administered in the form of suspension in DAE 433. The appropriate suspensions of the test item (50%, 5%, 0.5% w/v) was applied to three animals in volume 25 µl to the dorsum of each ear once a day morning for 3 consecutive days. During the pilot experiment no clinical symptoms of systemic toxicity were observed. In treated animals no erythema and skin reaction were observed. MAIN STUDYANIMAL ASSIGNMENT AND TREATMENTAnimals were subjected to a clinical examination (health check) shortly after arrival. No clinical changes were recorded.After acclimatization the animals have been randomly allocated to the dose groups and assigned animal numbersTREATMENT PREPARATION AND ADMINISTRATION:Dosage volume: 25 µl /ear/animalThe application forms of test item (suspension) were prepared immediately before administration.IN VIVO EXAMINATION-mortality- clinical observation- body weightPOST MORTEM INVESTIGATIONS- ears weights- Incorporation of 3H-methyl Thymidine

Positive control substance(s):

other: Dinitrochlorobenzene (DNCB)

Statistics:

For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. Statistical evaluation of measured parameters was performed at first by applying the non-parametric Kruskal-Wallis test for testing whether all group samples originate from the same distribution, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) for two-group comparisons.

Results and discussion

Positive control results:

The positive control substance DNCB produced positive LLNA response at an exposure level expected to give an increase in the Stimulation Index SI ≥ 3 over the negative control group, which was in congruence with the expected mode of action of a contact allergen. The positive control also elicited a reaction pattern with statistically significant increase in ear weight. These results demonstrate that the method performed in conditions of our laboratory has sufficient reliability.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

< 3

Test group / Remarks:

all test group

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The value of DPM at all dose levels is not statistically significantly increased compared to negative control.

Any other information on results incl. tables

Summary table of results

Group	Radioisotope incorporation		Ear weight
	Mean DPM	SI	Mean (mg)
NC	267.22	1.00	24.18
PC	2289.34	8.57*+	35.00*
50%	265.70	0.99	23.74
5%	287.49	1.08	23.80
0.5%	305.96	1.14	22.92*

Notes: SI - stimulation index

DPM -disintegrations per minute

 Figures with asterisk * = values statistically significant on probability level < 0.05

(Mann-Whitney test)

Figures with cross+= values ≥ 3 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the given test conditions, the animals exposed to the tested concentrations of the test item, Akardit, does not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that the test substance Akardit could not be a contact allergen in mice. The test item did not caused a statistically significant increase in radioisotope incorporation into the DNA of dividing lymphocytes so the test item, Akardit, provides negative sensitising response in LLNA assay.

Executive summary:

The test item, Akardit, was tested for the assessment of skin sensitisation potential with the murine local lymph node assay. This study is a part of the test item health hazard evaluation.

 

The Local Lymph Node Assay (LLNA) with radionuclide was used. The testing was conducted according totheMethod B.42 – Skin Sensitisation: Local Lymph Node Assay, Council Regulation (EC) No.640/2012, published in O.J. L 193, 2012

 

In this study the contact allergenic potential of Akardit was evaluated after topical application to female BALB/c mice. Mice were exposed to three concentrations of test item suspended in vehicle (DAE 433) for 3 consecutive days.

In pilot experiment the following concentrationsof test item in application forms were used:50 %, 5 %, 0.5 % (w/v). According to the results of pilot experiment the doses were confirmed for main study.

 

 Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated using radioactive labelling of proliferating cells. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, was determined. The evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

 

Comparison of Stimulation Indexes between treated groups and control vehicle group revealed that the test itemAkarditdid not cause a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes. The Stimulation Index of all treated groups is < 3 and the value of DPMis not statistically significantly increased compared to negative control.

The test item Akardit did not cause statistically significant increase of ear weight and irritation to skin at all dose level – it means the test item did not cause irritation to skin.

 

The animals exposed to the test item at all concentrations showed no pathological and no other negative clinical symptoms of intoxication throughout the experiment.

 

The positive control item DNCB (concentration 0.5% (w/v) elicited a reaction pattern with statistically significant increase in Stimulation Index of cell proliferation and of ear weight, which was in congruence with his expected mode of action as a contact allergen. Appropriate performance of the assay in the test laboratory was then demonstrated.

 

Under the given test conditions,the test item, Akardit, provides negative sensitising response in LLNA assay.