4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 26 April 2000 Experimental completion date: 2 May 2000.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar 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:

4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

EC Number:

260-152-0

EC Name:

4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

Cas Number:

56396-10-2

Molecular formula:

C25H20N4O4

IUPAC Name:

4-[[5-(anilino)carbonyl-2-methoxyphenyl]azo]-3-hydroxynaphthalene-2-carboxamide

additive 1

Reference substance name:

Calcium abietate

EC Number:

236-677-6

EC Name:

Calcium abietate

Cas Number:

13463-98-4

Molecular formula:

C20H30O2.1/2Ca

IUPAC Name:

calcium bis((1R,4aR,4bR,10aR)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,4b,5,6,10,10a-decahydrophenanthrene-1-carboxylate)

Test material form:

solid: particulate/powder

Details on test material:

An additive (CAS 13463-98-4) was present in the sample, at 10 % w/v

Specific details on test material used for the study:

Identity: ST383
Appearance: Magenta powder
Storage conditions: Room temperature
Batch number: S/No 8793
Expiry: 13 October 2000 (6 months from receipt)
Composition: Magenta pigment
Date received: 13 April 2000

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Sixteen healthy female CBA/Ca mice were obtained from Harlan UK Ltd., Oxon, England on 20 April 2000.
Animals were within the weight range 15.5 to 18.9g and approximately six to eight weeks of age prior to dosing on Day 1. All the mice were acclimatised to the experimental environment for six days prior to allocation to the test.
The mice were allocated without conscious bias to cages within the treatment groups. They were housed in groups of four mice in plastic cages with sawdust bedding.
A standard laboratoiy rodent diet (Special Diet Services RM1 (E) SQC) and drinking water were provided ad libitum.
The animal room environmental parameters were continuously recorded using a seven day chart recorder. The temperature was controlled within the range 20 to 21.5°C and relative humidity within the range 43 to 51%. Lighting was controlled by means of a time switch to give 12 hours of artificial
light (0600 - 1800 hours GMT) in each 24 hour period.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

5, 10 and 20% w/v in vehicle AOO

No. of animals per dose:

4 mice for each concentration.

Details on study design:

TEST SUBSTANCE PREPARATION
The test substance was prepared in the vehicle (AOO ) at the required concentrations freshly on each day of dosing .
The absorption of the test substance was not determined.


TREATMENT PROCEDURE:
Administration of the test substance:
Groups of four mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 µL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days . The test substance was applied to the dorsal surface of each ear using an automatic micropipette. The test substance was spread over the entire dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.

Selection of dosage levels:
A vehicle trial indicated that 20% w/v in AOO was the maximum practical concentration, therefore the following concentrations were selected:
5, 10 and 20% w/v

Administration of 3H-methyl Thymidine:
Five days following the first topical application of test substance all mice were injected via the tail vein with 250 µL of physiological saline containing 3H-methyl thymidine (3 HTdR: 80 µCi /ml) giving a nominal 20 µCi to each mouse. The injection into the tail vein was carried out with the mice restrained in a hot box, using a plastic syringe and needle.

OBSERVATIONS
Clinical signs:
All animals were observed daily for signs of ill health or toxicity. The ears were also examined for signs of irritation.
Bodyweight:
The weight of each mouse was recorded on arrival, Day 1 (first day of dosing) and prior to termination (Day 6).

TERMINAL STUDIES
Termination:
Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation and the draining auricular lymph nodes excised and pooled for each experimental group. 1.0 ml of physiological saline was added to the pooled lymph nodes for each group. The animals were then discarded and no further investigations were carried out.

Preparation of single cell suspensions:
A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size) using the plunger of a syringe. The pooled LNC were then washed by adding 10 ml physiological saline, pelleted at 190 g for 10 minutes and resuspended. The cells were washed three times and resuspended in trichloroacetic acid (TCA: 5%) following the final wash.

Determination of incorporated 3H-methyl thymidine:
After overnight incubation with 5% TCA at 4°C, the precipitate was recovered by centrifugation and resuspended in 1 ml 5% TCA and transferred to 10 ml scintillation fluid. 3HTdR incorporation was measured by (beta-scintillation counting. The proliferative response of LNC was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio).

INTERPRETATION OF RESULTS
The test substance is regarded as a sensitiser if at least one concentration of the test substance results in
a three-fold greater increase in 3HTdR incorporation compared to control values.

Positive control substance(s):

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

Results and discussion

Positive control results:

Groups of four mice were used in this study. Three dosage levels and a vehicle control were used with dosages of HCA selected as follows:
10, 25, 50% v/v in AOO (4:1 v/v acetone/olive oil).

The positive control study is considered to be valid if at least one concentration of HCA results in a three-fold or greater increase in 3HTdR incorporation compared to control values.

Results:
The test/control ratios obtained for 10, 25 and 50% v/v HCA were 1.8, 3.3 and 3.4 respectively. This indicates that HCA showed the potential to induce skin sensitisation (delayed contact hypersensitivity) and confirms the sensitivity of the technique to detect skin sensitization.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

test/ control ratio

Value:

1.9

Test group / Remarks:

5 % w/v

Remarks on result:

other: negative for skin sensitisation

Results 2

Key result

Parameter:

SI

Remarks:

test/ control ratio

Value:

1.2

Test group / Remarks:

10 % w/v

Remarks on result:

other: negative for skin sensitisation

Results 3

Key result

Parameter:

SI

Remarks:

test/ control ratio

Value:

1.1

Test group / Remarks:

20 % w/v

Remarks on result:

other: negative for skin sensitisation

Cellular proliferation data / Observations:

CLINICAL SIGNS
No signs of ill health or toxicity were observed. Red staining behind the ears was noted for all groups receiving the test material.

BODYWEIGHT
Bodyweight increases were recorded for all mice over the period of the study.

ESTIMATION OF THE PROLIFERATIVE RESPONSE OF LYMPH NODE CELLS
The test/control ratios obtained for 5, 10 and 20 % w/v ST383 were 1.9, 1.2 and 1.1 respectively. As a test/control ratio of greater than 3 was not recorded for any of the concentrations tested, ST383 is not considered to have the potential to cause skin sensitization (delayed contact hypersensitivity).

Any other information on results incl. tables

Group dpm/node and test/control ratios

Group	Concentation % w/v	dpm	number of lymph nodes	dpm/node	test/control ratio*	+ = positive  - = negative
1	Control	5178.1	8	647.3	na	na
2	5	9804.8	8	1225.6	1.9	-
3	10	6201.1	8	775.1	1.2	-
4	20	5649.8	8	706.2	1.1	-

Control/vehicle = AOO

* Test/control ratio of 3 or greater indicates a positive result

na = Not applicable

dpm = disintegrations per minute

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

ST383 is not regarded as a sensitizer

Executive summary:

The study was performed to assess the skin sensitization potential of ST383 using the murine local lymph node assay (LLNA). The assay can be used as a first stage assessment of skin sensitisation potential as indicated in the OECD Guideline for Testing of Chemicals No. 406 “ Skin Sensitisation” Adopted 17 July 1992 and EEC Methods for the determination of toxicity, Annex to Directive 96/54/EC (Official Journal No. L248, 30.9.96), Part B, Method B.6. Skin sensitisation.

Groups of four mice were used in this study at three dosage levels and a vehicle control. Dosages were selected as follows:

5, 10 and 20% w/v ST383 in AOO (4:1 v/v acetone/olive oil)

Each group of mice was treated by daily application of 25 µL of each of one of these three concentrations, and vehicle control, to the dorsal surface of both ears for three consecutive days.

The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of ³H-Thymidine by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of ³HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio).

The test substance is regarded as a sensitiser if at least one concentration of the chemical results in a three-fold greater increase in ³HTdR incorporation compared to control values.

In this assay the test/control ratios obtained for 5, 10 and 20% w/v are 1.9, 1.2 and 1.1 respectively which indicates that ST383 did not show the potential to induce skin sensitisation (delayed contact hypersensitivity).

Responses to the positive control substances hexyl cinnamic aldehyde (HCA) in a positive control study demonstrate the reliability and sensitivity of this assay.

Conclusion

ST383 is not regarded as a sensitiser.