Butyl 2-[[3-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-hydroxy-1-naphthyl]azo]benzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD TG 429), performed under GLP

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:

Butyl 2-[[3-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-hydroxy-1-naphthyl]azo]benzoate

EC Number:

250-800-0

EC Name:

Butyl 2-[[3-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-hydroxy-1-naphthyl]azo]benzoate

Cas Number:

31778-10-6

Molecular formula:

C29H25N5O5

IUPAC Name:

butyl 2-({2-hydroxy-3-[(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)carbamoyl]-1-naphthyl}diazenyl)benzoate

Test material form:

solid: nanoform, no surface treatment

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 (%): 20-70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0, 3, 6, and 12% (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 as solvent because it was the most suitable solvent
- 12% (w/v) was the highest technically applicable concentration
- Irritation: no signs of systemic toxicity after single application of test item concentration of 12% (w/v), no signs of irritation after single application of test item concentrations of 1.5, 3, 6 or 12% (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
- 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 on 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 2.0, 3.0 and 4.9 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

Parameter:

SI

Value:

ca. 1.3

Test group / Remarks:

TG 4-5 Concentration 12%

Results 2

Parameter:

SI

Value:

ca. 1.15

Test group / Remarks:

TG 3-5Concentration 6%

Results 3

Parameter:

SI

Value:

ca. 0.96

Test group / Remarks:

TG2-5 Concentration 3%

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Background of measurement: 5.7 - 0.2; mean: 2.95 (was taken for calculation) DPM per lymph node control group: 1622.8 3% test item: 1559.1 6% test item: 1863.6 12% test item: 2105.8

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 fades) a)	S.l.*	number of lymph nodes		S.I.
---	BG I	5.7	---	---	---	---	---
---	BG II	0.2	---	---	---	---	---
---	CG 1_1	3403.7	3400.8	---	---	---	---
---	CG 1_2	2277.6	2274.7	---	---	---	---
---	CG 1_3	5040.7	5037.8	---	---	---	---
---	CG 1_4	2706.6	2703.7	---	---	---	---
---	CG 1_5	2814.4	2811.5	---	10	1622.8	1.00
3	TG 2_1	4139.7	4136.8	1.3	---	---	---
3	TG 2_2	3227.1	3224.2	1.0	---	---	---
3	TG 2_3	1910.6	1907.7	0.6	---	---	---
3	TG 2_4	3719.6	3716.7	1.1	---	---	---
3	TG 2_5	2608.4	2605.5	0.8	10	1559.1	0.96
6	TG 3_1	2513.0	2510.1	0.8	---	---	---
6	TG 3_2	4114.2	4111.3	1.3	---	---
6	TG 3_3	3077.9	3075.0	0.9	---	---	---
6	TG 3_4	3812.6	3809.7	1.2	---	---	---
6	TG 3_5	5133.3	5130.4	1.6	10	1863.6	1.15
12	TG 4_1	4933.9	4931.0	1.5	---	---	---
12	TG 4_2	4677.2	4674.3	1.4	---	---
12	TG 4_3	4723.4	4720.5	1.5	---	---	---
12	TG 4_4	3793.0	3790.1	1.2	---	---	---
12	TG 4_5	2945.1	2942.2	0.9	10	2105.8	1.30

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

CG = Control Group

TG = Test Group

S I = Stimulation Index

=mean net DPM value per lymph node of the mause divided by the mean net DPM value of the vehicle control group

a)                                    The mean value was taken from the figures BG 1 and BG II

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Tested in this study according to OECD test guideline no. 429 and under GLP conditions the test item was not a skin sensitizer. The results were verified in a second, independent, non-GLP Local lymph node assay.

Executive summary:

In the study the test item dissolved 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 3.0, 6.0, and 12.0 %.

No cases of mortality were observed. Due to the intense red colour of the test item local irritation reactions such as ear redness could not be detected. No swelling of the ears was observed.

In this study Stimulation Indices (S.I.) of 0.96, 1.15, and 1.30 were determined with the test item at concentrations of 3.0, 6.0, and 12.0 % (w/v) in DMSO, respectively.

To verify the results of the assay, a repeat experiment was performed under non-GLP conditions using 12 % (w/v) in DMSO (4 CBA female mice/group; control and treatment group).

In the repeat experiment a Stimulation Index (S.I.) of 2.26 was calculated. The animals did not show any clinical signs and no mortality occurred during the course of the study.

The test item was not a skin sensitiser in the assays.