5-(benzoylamino)-3-[[5-[[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]methyl]-1-sulpho-2-naphthyl]azo]-4-hydroxynaphthalene-2,7-disulphonic acid, lithium sodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989-04-04 to 1990-01-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted: 12th May 1981

Deviations:

no

GLP compliance:

no

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Existing data from 1990

Test material

Test material information

Constituent 1

Reference substance name:

5-(benzoylamino)-3-[[5-[[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]methyl]-1-sulpho-2-naphthyl]azo]-4-hydroxynaphthalene-2,7-disulphonic acid, lithium sodium salt

EC Number:

280-427-9

EC Name:

5-(benzoylamino)-3-[[5-[[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]methyl]-1-sulpho-2-naphthyl]azo]-4-hydroxynaphthalene-2,7-disulphonic acid, lithium sodium salt

Cas Number:

83400-12-8

Molecular formula:

C32 H21 Cl F2 N6 O11 S3 . x Li . x Na C32H(21-x-y)ClF2Li(x)N6Na(y)O11S3

IUPAC Name:

5-(benzoylamino)-3-[[5-[[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]methyl]-1-sulpho-2-naphthyl]azo]-4-hydroxynaphthalene-2,7-disulphonic acid, lithium sodium salt

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Versuchstierzucht Winkelmann, Borchen, Germany
- Microbiological status of animals, when known: SPF
- Age at study initiation: 4 -7 weeks
- Weight at study initiation: Mean: 319 g (from 280 to 363 g)
- Housing: Animals (5 animals per cage) were housed in type IV Makrolon® cages with low-dust granula (Ssniff Spezialdiäten GmbH, Soest, Germany). The animal room was cleaned once weekly and disinfected once per month. Contamination of the feed and the animals was excluded.
- Diet (e.g. ad libitum): Altromin 3022, ad libitum
- Water (e.g. ad libitum): Tap water; ad libitum
- Acclimation period: at least 7 days
- Indication of any skin lesions: Only healthy, symptom-free animals were used

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

2.5% in physiological saline

Day(s)/duration:

Day 1 - single application

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

25% in physiological saline

Day(s)/duration:

one week after intradermal induction/48 hours

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Challenge

Challenge 1

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

Concentration: 25%
Amount: 0.5 mL

Day(s)/duration:

three weeks after intradermal induction/24 hours

Adequacy of challenge:

highest non-irritant concentration

Challenge 2

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

Concentration: 2.5 and 12%
Amount: 0.5 mL

Day(s)/duration:

four weeks after intradermal nduction/24 hours

Adequacy of challenge:

not specified

No. of animals per dose:

20 guinea pigs in the test group, 10 in the control group

Details on study design:

RANGE FINDING TESTS:
One guinea pig received intradermally 0.1 mL of the following test item concentrations: 0, 1, 2.5 and 5 %. The injection sites were assessed 24 and 48 h after application. For results please refer to box "Any other information on results incl. tables".
- Epicutaneous application:
4 guinea pigs recieved under occlusive conditions each of the following test item concentrations: 3%, 6%, 12% and 25% for 24 h. The application sites were assessed after 48 and 72 h.
For results please refer to box "Any other information on results incl. tables".

MAIN STUDY
A.INDUCTION EXPOSURE
Intradermal application
- No. of exposures: 1
- Test groups:
Three pairs of intradermal injections of 0.1 mL volume were given in the shoulder region so that one of each pair lies on each side of the midline.
Injection 1 (cranial): a 1:1 mixture (v/v) FCA/ physiological saline
Injection 2 (medial): 2.5% suspension of the test material in physiological saline
Injection 3 (caudal): 2.5% suspension of the test material formulated in a 1:1 mixture (v/v) FCA/ physiological saline.
- Control group:
Three pairs of intradermal injections of 0.1 mL volume were given in the shoulder region so that one of each pair lies on each side of the midline.
Injection 1 (cranial): a 1:1 mixture (v/v) FCA/ physiological saline
Injection 2 (medial): physiological saline
Injection 3 (caudal): physiological saline in a 1:1 mixture (v/v) FCA/ physiological saline.
- Frequency of applications: 1

Epicutaneous application
- No. of exposures: 1
- Exposure period: 48 h
One day before exposure, the application sites have been clipped and coated with 0.2 mL of a 10% sodium lauryl sulfate solution in paraffin oil. A hypoallergenic band-aid (2 x 4 cm) impregnated with 0.5 mL of the test suspension (25 % test material in physiological saline (test group) or physiological saline (control)) was put on the injection sites of the intradermal application, covered with aluminium foil and fixed for 48 h using a Fermoflex tape (Transatlantic GmbH, Schwarzenbach). At the end of the exposure period the substance was removed using physiological saline.

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 21 and 28 days after intradermal induction
- Exposure period: 24 h
One day before exposure, the application sites have been clipped. Two hypoallergenic band-aids (2 x 4 cm), impregnated with 0.5 mL of the test item suspension (25% test material in physioloigical saline), were applied to the left flank of all animals (test and first control group) and fixed for 24 h using a Fermoflex tape (Transatlantic GmbH, Schwarzenbach).
For the second challenge, animals (test and second control group) have been applied two hypoallergenic band-aids impregnated with 0.5 mL the test suspension (12 and 2.5% test material in physioloigical saline) were applied to the left flank and fixed using a Fermoflex tape. In the two concentrations and alternating from animal to animal, the patches were applied cranially or caudally. On the right flank two control plaster were applied, soaked with physiological saline (0.5 mL application volume). At the end of the exposure time, application sites have been chemically depilated using Pilca-Crème (Olivin GmbH, Hamburg, Germany).
- Concentrations: 25% (1. Challenge, 21 days after intradermal induction), 12 and 2.5% (2. Challenge, 28 days after intradermal injection)
- Evaluation (hr after challenge): 48 and 72 h
- Scoring: The skin reaction have been scored as proposed by Magnusson and Kligman (see Table 1 in box "Any other information on material and methods incl. tables")

Challenge controls:

10 animals previously treated with the vehicle were challenged together with the test group using the same test article concentration as for the test group.

Positive control substance(s):

no

Results and discussion

Positive control results:

n.a.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

11

Total no. in group:

20

Clinical observations:

erythema

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

25%

No. with + reactions:

9

Total no. in group:

20

Clinical observations:

erythema

Remarks on result:

positive indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Results 4

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Results 5

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

12%

No. with + reactions:

14

Total no. in group:

20

Clinical observations:

erythema

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

rechallenge

Hours after challenge:

72

Group:

test chemical

Dose level:

12%

No. with + reactions:

6

Total no. in group:

20

Clinical observations:

erythema

Remarks on result:

positive indication of skin sensitisation

Results 7

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

12%

No. with + reactions:

0

Total no. in group:

10

Results 8

Reading:

rechallenge

Hours after challenge:

72

Group:

negative control

Dose level:

12%

No. with + reactions:

0

Total no. in group:

10

Results 9

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

2.5%

No. with + reactions:

8

Total no. in group:

20

Clinical observations:

erythema

Remarks on result:

positive indication of skin sensitisation

Results 10

Reading:

rechallenge

Hours after challenge:

72

Group:

test chemical

Dose level:

2.5%

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

erythema

Remarks on result:

positive indication of skin sensitisation

Results 11

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

2.5%

No. with + reactions:

0

Total no. in group:

10

Results 12

Reading:

rechallenge

Hours after challenge:

72

Group:

negative control

Dose level:

2.5%

No. with + reactions:

0

Total no. in group:

10

Results 13

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0%

No. with + reactions:

0

Total no. in group:

20

Results 14

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

0%

No. with + reactions:

0

Total no. in group:

20

Any other information on results incl. tables

Results of the pre-test

- Intradermal induction

After 24 and 48 hours the area around the injection site was stained red. After 48 hours, the 5% injection site was stained dark red.

 

- Topical induction

Table 2: Results for skin reaction after topical application in the pre-test

Animal No.	Test material concentration
	3%		6%		12%		25%
	48 h	72 h	48 h	72 h	48 h	72 h	48 h	72 h
2	0	0	0	0	0	1	0	1
3	0	0	0	0	0	0	0	0
4	0	0	0	0	0	0	0	0
5	0	0	0	0	0	0	0	0

Results of the main test

Table 3: Individual results of the 1. Challenge

Animal No.	Test material concentration
	25%
	48 h	72 h
1. Control group
1	0	0
2	0	0
3	0	0
4	0	0
5	0	0
6	0	0
7	0	0
8	0	0
9	0	0
10	0	0
Test group
21	0	0
22	1	1
23	1	1
24	1	1
25	0	0
26	0	0
27	1	1
28	0	0
29	0	0
30	1	1
31	0*	0*
32	1	1
33	0*	0*
34	0*	0*
35	1	0
36	1	1
37	1	1
38	1	0
39	0	0
40	1	1

* = Application site was stained blue violet

  

Table 4: Individual results of the 2. Challenge

Animal No.	Test material concentration
	12%		2.5%
	48 h	72 h	48 h	72 h
1. Control group
11	0	0	0	0
12	0	0	0	0
13	0	0	0	0
14	0	0	0	0
15	0	0	0	0
16	0	0	0	0
17	0	0	0	0
18	0	0	0	0
19	0	0	0	0
20	0	0	0	0
Test group
21	1	1	0	0
22	2	1	1	1
23	1	0	0	0
24	2	1	0	0
25	0	0	0	0
26	1	0	1	0
27	2	1	1	0
28	1	0	0	0
29	0	0	0	0
30	1	1	1	0
31	1	0	0	0
32	0	0	0	0
33	2	1	0	0
34	1	0	1	1
35	1	0	1	0
36	0	0	0	0
37	1	0	1	0
38	0	0	0	0
39	0	0	0	0
40	1	0	1	0

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

In conclusion, based on the results obtained from a dermal skin sensitisation test, the test item can be considered as a dermal sensitizer.

Executive summary:

In a dermal sensitization study conducted according to OECD 406, young male Bor:DHPW guinea pigs (20 animals/test group & 10 animals/control) were tested using the Guinea Pig Maximisation Test according to Magnusson and Kligman. The animals received intradermally 2.5% of the test item in physiological saline. One week after the intradermal induction, the animals received the second induction by applying the test item as a 25% suspension in physiological saline topically. Three weeks after the intradermal induction, the animals were challenged with a 25% suspension in physiological saline of the test item. The animals of the control group were not treated during the induction, but were treated once at each challenge. After the first challenge the treatment group showed erythema at the application site. During a second challenge the animals were treated with either 12% or 2.5% suspensions in physiological saline. During the re-challenge, erythema was again observed only in the treatment groups. No effects were observed in the control group animals. Therefore, based on the results obtained, the test item can be considered as a dermal sensitizer.