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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 OCT 2004 to 10 Nov 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:

2005

Report date:

2005

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:

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

EC Number:

250-830-4

EC Name:

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

Cas Number:

31837-42-0

Molecular formula:

C18H15N5O5

IUPAC Name:

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

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 I cages with wire mesh top and granulated sooft wood bedding
- Diet: Pelleted standard diet (provided by Harlan Winkelmann GmbH, Borchen, Germany); ad libitum
- Water: Community tap water from Rossdorf, ad libitum
- Acclimation period: yes, but duration not mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0, 3.75, 7, and 15% (w/v)

No. of animals per dose:

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

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: DMSO was selected because in any other vehicle test item concentrations above 15% could not be obtained or due to the instability of the test item withinh the vehicle
- 15% (w/v) was the highest technically applicable concentration
- Irritation: no systemic toxicity after single application of test item concentrations up to 15% (w/v); irritating effects could not be assessed due to yellow staining at concentration of 15% (w/v), but no ear swelling was observed
- 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
- 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
- A statistical analysis was conducted for assessment of the dose-response relationship, and 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 an the local lymph node assay dose response Plot.
- The Dunnett-test (ANOVA) was conducted to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. Statistical significance was at the five per cent level (p < 0.05).

Results and discussion

Positive control results:

Stimulation indices of 1.96, 3.03 and 4.92 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 (= 9.9% (w/v)).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

>= 1.4 - <= 1.6

Test group / Remarks:

all

Remarks on result:

other: Stimulation indices (SI) were all below 3. The following SI were calculated: 3.75% test item: 1.4 7% test item: 1.3 15% test item: 1.6

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM/lymph node Background of measurement: 0 - 3.4; mean: 1.7 (was taken for calculations) control group: 613.4 3.75% test item: 868.4 7% test item: 872.1 15% test item: 1026.9

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		Measurement DPM	Calculation	Result	Calculation	DPM per lymph node b)	Result
% (w/v)	Group		DPM-BG per animal (2 lymph nodes) a)	S.I. *	number of lymph nodes		S.1
-	BG I	3.4		---			--
	BG II	0,0	--- ---	---	-	--	--
	CG 1_1	891.4	889,7				---
-	CG 1_2	980.9	979.2	---	--
	CG 1_3	1392.9	1391.2		---	---	--
---	CG 1_4	1378.5	1376,8		-	-
	CG 1_5	1679.0	1677.3		10	631.4	1.0
3.75	TG 2_1	2339.5	2337.8	1,9
3.75	TG 2_2	1481.9	1480.2	1,2			--
3.75	TG 2_3	1498.5	1496.8	1.2			--
3.75	TG 2_4	1743.1	1741.4	1.4	---	-	--
3.75	TG 2_5	1629.6	1627.9	1.3	10	868.4	1.4
7.0	TG 3_1	1140.0	1138.3	0.9			--
7.0	TG 3_2	1822.8	1821.1	1.4		---
7.0	TG 3_3	1665.0	1663.3	1.3	---
7.0	TG 3_4	2168.6	2166.9	1.7	---	---	--
7.0	TG 3_5	1483.4	1481.7	1.2	10	827.1	1.3
15.0	TG 41	2075.9	2074.2	1.6			--
15.0	TG 4_2	1870.6	1868.9	1.5	---	-
15.0	TG 4_3	2893.5	2891.8	2,3
15.0	TG 4_4	1936.7	1935.0	1.5
15.0	TG 4_5	1501.0	1499.3	1.2	10	1026.9	1.6

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

CG = Control Group

TG = Test Group

S.l. = Stimulation Index

* = mean net DPM value per lymph node of the mouse divided by the mean net DPMvalueof the vehiclecontrol group

a)        = The mean of the background value (BGI and BGH) was subtracted from the value.

b)        = DPM/node was determined by dividing the sum of the measured values from all lymph nodes within a
group by the number of lymph nodes taken from that group

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Criteria used for interpretation of results: EU

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 (LLNA) was performed using test item concentrationsof 3.75 %, 7.0 %, and 15.0 % (w/v).

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 1.4, 1.3, and 1.6 were determined with the test item at concentrations of 3.75 %, 7.0 %, and 15.0 % (w/v) in DMSO.

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