4-[(2,5-dichlorophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxynaphthalene-2-carboxamide

Endpoint summary

Description of key information

PB25

Tested in this study according to OECD test guideline no. 429 and GLP conditions.For this purpose a local lymph node assay was performed.In the study the test item dissolved in acetone:olive oil, 4:1 (v/v) was assessed for its possible contact allergenic potential. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. The test item was not a skin sensitiser in this assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

DEC 2004 to 25 JAN 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

due to technical reasons only one lymph node could be analysed for one animal

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

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

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 3.75, 7.5, 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: acetone/olive oil (4:1) was selected as solvent because it was the most suitable solvent
- 15% (w/v) was the highest technically applicable concentration
- Irritation: no signs of systemic toxicity after single application of test item concentration of 15% (w/v). Due to the intense brown colour of the test item local irritation reactions such as ear redness could not be detected. No swelling of the ears 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 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).

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)).

Parameter:

SI

Value:

1

Test group / Remarks:

CG 1_5

Remarks on result:

other: Stimulation indices (SI) were all below 3. The following SI were calculated: 3.75% test item: 1.35 7.5% test item: 0.73 15% test item: 0.79

Parameter:

SI

Value:

1.35

Test group / Remarks:

TG 2_5

Remarks on result:

other: Stimulation indices (SI) were all below 3. The following SI were calculated: 3.75% test item: 1.35 7.5% test item: 0.73 15% test item: 0.79

Parameter:

SI

Value:

0.73

Test group / Remarks:

TG 3_5

Remarks on result:

other: Stimulation indices (SI) were all below 3. The following SI were calculated: 3.75% test item: 1.35 7.5% test item: 0.73 15% test item: 0.79

Parameter:

SI

Value:

0.79

Test group / Remarks:

TG 4_5

Remarks on result:

other: Stimulation indices (SI) were all below 3. The following SI were calculated: 3.75% test item: 1.35 7.5% test item: 0.73 15% test item: 0.79

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.I.
---	BG I	2.9	---	---	---	---	----
---	BG II	3.7	---	---	---	---	---
---	CG 1_1	1905.2	1901.9	---	---	---	---
---	CG 1_2	1269.1	1265.8	---	---	---	---
---	CG 1_3	1384.1	1380.8	---	---	---	---
---	CG 1_4	1522.3	1519.0	---	---	---	---
---	CG 1_5	1667.7	1664.4	---	10	773.2	1.00
3.75	TG 2_1	1466.3	1463.0	0.9	---	---	---
3.75	TG 2_2	2294.1	2290.8	1.5	---	---	---
3.75	TG 2_3	2562.7	2559.4	1.7	---	---	---
3.75	TG 2_4	1941.8	1938.5	1.3	---	---	---
3.75	TG 2_5	1112.5	1109.2^c)	0.7	9	1040.1	1.35
7.5	TG 3_1	823.7	820.4	0.5	---	---	---
7.5	TG 3_2	1316.3	1313.0	0.8	---	---	---
7.5	TG 3_3	1252.3	1249.0	0.8	---	---	---
7.5	TG 3_4	984.3	981.0	0.6	--	---	---
7.5	TG 3_5	1305.8	1302.5	0.8	10	566.6	0.73^s
15.0	TG 4_1	1113.7	1110.4	0.7	---	---	---
15.0	TG 4_2	898.2	894.9	0.6	---	---	---
15.0	TG 4_3	1420.1	1416.8	0.9	---	---	---
15.0	TG 4_4	1352.7	1349.4	0.9	---	---	---
15.0	TG 4_5	1313.8	1310.5	0.8	10	608.2	0.79

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

CG = Control Group

TG = Test Group

5.1 = Stimulation Index

* = mean net DPM value per lymph node of the mouse divided by the mean net DPM value of the vehicle control 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

c)                  DPM value of one lymph node

s = Mean DPM value for the group was according to the Dunnett test significantly lower than corresponding control value. The p value for the analysis was 0.038. This is, however,irrelevant, since the significant value (0.73) is below the control value and not above.

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

In the study the test item dissolved in acetone:olive oil, 4:1 (v/v)was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 0, 3.75, 7.5 and 15 % (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.l..) of 1.35, 0.73 and 0.79 were determined with the test item at concentrations of 3.75, 7.5 and 15 % (w/v) in acetone:olive oil, 4:1 (v/v), respectively,

The test item was not a skin sensitiser in this assay

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available