1,3-dimethyl-1,3-diphenylurea

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17.08.-30.08.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

1,3-dimethyl-1,3-diphenylurea

EC Number:

210-283-4

EC Name:

1,3-dimethyl-1,3-diphenylurea

Cas Number:

611-92-7

Molecular formula:

C15H16N2O

IUPAC Name:

1,3-dimethyl-1,3-diphenylurea

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

EXPERIMENTAL ANIMALS
Animals species and strain: Young adult female mice (nulliparous and non-pregnant), strain BALB/CBYJICO (referred to as BALB/c in this document)
Source: Breeding farm VELAZ s.r.o., Unětice 139, 252 62, Czech Republic, RČH CZ 21760118
Number of animals per group

Age: 8 to 10 weeks (at start of dosing)
Body weight range: 16.13 - 19.89 g (at start of dosing), in pilot experiment 16.73 – 18.06 g
Health examination: All animals were examined during the acclimatisation period
Acclimatisation: 21 days

Animal quarters/husbandry
Animal rooms: Monitored conditions, microbiologically defined background, according to internal SOP No.40
Microclimatic conditions:
- Room temperature: 22 ± 3 °C, permanently monitored
- Relative humidity: 30 – 70 %, permanently monitored
- Light: 12 hours light/dark cycle: 6am-6pm/6pm-6am


Animal caging: Animals in groups in macrolon cages with sterilized softwood shavings
Water: Drinking tap water ad libitum. Water quality corresponded to Regulation No. 252/2004 Czech Coll. Of Law, Health Ministry
Diet: Pelleted standard diet for experimental animals ad libitum (Altromin, manufacturer: Altromin Spezialfutter GmbH & Co. KG, Germany). Microbiological control and content of nutrients is performed according internal SOP No. 72.
Prophylactic arrangement: Cleaning and disinfection of animal room was regularly performed, as it is described in internal SOP No.10.

Animal identification
Cage identification: By cage number, study number and group specific colour.
Animal identification: By felt tip marking (from 1 to 5 per each group and group specific colour)

Study design: in vivo (LLNA)

Vehicle:

other: DAE 433

Concentration:

DAE 433 – mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol

Vehicle selection justification
The vehicle DAE 433- This vehicle is used in our laboratory for approximately 10 years and it elicited a consistent response over whole period.

No. of animals per dose:

Pilot experiment – 3 females
Exposed groups – 15 females (5 animals in three groups)
Positive control group – 5 females
Negative control group – 5 females


Total: 28 animals

Positive control substance(s):

other: Dinitrochlorobenzene (DNCB)

Results and discussion

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

< 3

Test group / Remarks:

Pilot experiment – 3 females, Exposed groups – 15 females (5 animals in three groups), Positive control group – 5 females, Negative control group – 5 females, Total: 28 animals

Cellular proliferation data / Observations:

The value of DPM and SI for positive control group was increased. The SI was ≥ 3 (17.12) – the LLNA was efficient (see Table 9).
The SI for the test groups treated with the test substance at all dose level is below the threshold, and stimulation index (SI) is < 3 (see Table 9).
The value of DPM at all dose levels was not statistically significantly increased compared to negative control. Only slightly decrease of DPM value at the highest dose level was observed.

Any other information on results incl. tables

Table 9. Individual Activities and Calculated Values  

Group (Anim.No.)	NC (1-5)	PC (6-10)	50% (11-15)	5% (16-20)	0.5% (21-25)
Activity (DPM)	179.71	2446.81	187.93	168.97	193.75
	221.02	2969.63	152.25	233.50	169.47
	160.03	3801.41	160.53	133.78	160.82
	162.71	2861.19	158.09	129.68	151.46
	161.17	3070.35	122.53	146.98	137.47
mean	176.93	3029.88*	156.27	162.58	162.59
SD	25.92	492.18	23.34	42.50	21.07
SI	1.00	17.12	0.88	0.92	0.92

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the given test conditions, the animals exposed to the test substance, Methylcentralit, does not elicit sensitising response in LLNA assay. Negative results in cell proliferation revealed that the test substance Methylcentralit could not be a contact allergen in mice.
The test substance Methylcentralit, provides negative sensitising response in LLNA assay.

Executive summary:

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

 

The Local Lymph Node Assay (LLNA) with the incorporation of 3H-methyl thymidine 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 ofMethylcentralitwas evaluated after topical application to female BALB/c mice. Mice were exposed to three concentrations of test substance suspended in vehicle DAE 433 (mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol) for 3 consecutive days.

 

In pilot experiment the following concentrationsof test substance in application forms were used:50 %, 5 %, 0.5 % (w/v).According to the results of pilot experiment the same 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 substanceMethylcentralit did notcause a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes of animals treated by the test substance at all concentrations. The Stimulation Index of the treated groups is< 3 and the value ofdisintegrations per minute(DPM) is not significantly increased compared to negative control.

The test substance did not cause increase of ear weight or other indication of irritation to skin at all dose levels.

 

The animals exposed to the test substance at all doses showed no pathological and no other negative clinical symptoms of intoxication throughout the experiment, besides slightly weight loss in 4 animals at the highest dose level.

 

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

Under the given test conditions,the test substance, Methylcentralit, provides negative sensitising response in LLNA assay. The SI at all dose levels was <3.