Dimethyl 2-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azo]terephthalate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 4 FEB 2004 to 18 FEB 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD TG 429)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl 2-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azo]terephthalate

EC Number:

252-650-1

EC Name:

Dimethyl 2-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azo]terephthalate

Cas Number:

35636-63-6

Molecular formula:

C21H19N5O7

IUPAC Name:

dimethyl 2-({2-oxo-1-[(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)carbamoyl]propyl}diazenyl)terephthalate

Test material form:

solid: nanoform

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands BV, Horst, The Netherlands
- Age at study initiation: 8-12 weeks
- Housing: Individually in Macrolon Type 2 cages with standard sooft wood bedding
- Diet: Pelleted standard Provimi Kliba 3433 mouse maintenance diet (batch no. 78/03; provided by Provimi Kliba AG, CH-4303 Kaiseraugst); ad libitum
- Water: Community tap water from Itingen, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0, 2.5, 5, and 10% (w/v)

No. of animals per dose:

4 females per dose group
2 females in the pre-test

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: DMSO was selected because it was more suitable than any other vehicles
- 10% (w/v) was the highest technically applicable concentration
- Irritation: no severe local irritation nor any systemic toxicity after single application of test item concentrations up to 10% (w/v)
- Lymph node proliferation response: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
1: exposure to at least one concentration of the test item resulted in an incorporation of 3H-methyl thymidine at least 3-fold or greater than that recorded in control mice as indicated by the stimulation index
2: data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression

TREATMENT PREPARATION AND ADMINISTRATION:
- individual preparation of weight volume dilutions using a magnetic stirrer as homogenizer
- test item preparations were made feshly before each dosing occasion and no more than 4 hours prior to application to the ears
- 25 µl were spread over the entire dorsal surface of each ear lobe once daily for three consecutive days

Positive control substance(s):

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

Statistics:

- calculations of mean values and standard deviations for body weight
- the EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c; where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining Iymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the value of 3

Results and discussion

Positive control results:

Stimulation indices of 1.5, 2.3 and 8.4 were determined with the positive control item at concentrations of 5%, 10% and 25% (w/v), respectively, in acetone:olive oil, 4:1 (v/v). An EC3 value was calculated (= 11.7% (w/v)).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

< 3

Remarks on result:

other: Stimulation indices (SI) were all below 3. The following SI were calculated: 2.5% test item: 0.8 5% test item: 0.8 10% test item: 1.2

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Background of measurement: 2 - 2; mean: 2 (was taken for calculations) control group: 5461 2.5% test item: 4601 5% test item: 4270 10% test item: 6715

Results 3

Key result

Parameter:

SI

Value:

ca. 0.8

Test group / Remarks:

2.5%

Results 4

Key result

Parameter:

SI

Value:

ca. 0.8

Test group / Remarks:

5%

Results 5

Key result

Parameter:

SI

Value:

ca. 1.2

Test group / Remarks:

10%

Any other information on results incl. tables

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. Calculation of the EC3 value was not performed because no test concentration produced a SI of 3 or higher.

Test item concentration % (w/v)		Measurement dpm	Calculation			Result
			dpm - BG^a)	number of Iymph nodes	dpm per lymph node^b)	S.I.
--	BG I	2	--	--	--	-
--	BG II	2	--	--	--	-
--	CG 1	5461	5459	8	682	-
2.5	TG 2	4601	4599	8	575	0.8
5	TG 3	4270	4268	8	534	0.8
10	TG 4	6715	6713	8	839	1.2

BG = Background (1 ml 5 % trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

a)       =The mean value was taken from the figures BG I and BG II

b)       = Since the Iymph nodes of the animals of a dose group were pooled, DPM/node wasdetermined by dividing the measured value by the number of lymph nodes pooled

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Regulation (EC) no. 1272/2008

Conclusions:

Tested in this study according to OECD test guideline no. 429 and GLP conditions the test item was not a skin sensitizer.

Executive summary:

In the study the test item suspended in DMSO was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5 and 10%.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 0.8, 0.8 and 1.2 were determined with the test item at concentrations of 2.5, 5 and 10% (w/v) in DMSO, respectively.

The test item was not a skin sensitiser in this assay.