Sodium 4-[[4-[(4-hydroxy-2-methylphenyl)azo]phenyl]amino]-3-nitrobenzenesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

OECD Guidelines for Testing of Chemicals, Number 406 "Skin Sensitization", adopted by the Council on July 17, 1992 (reported Paris, April 29, 1993).

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

Directive 96/54/EEC, B.6. "Skin Sensitization", July 30, 1996.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study available is over 12 years old

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-[[4-[(4-hydroxy-2-methylphenyl)azo]phenyl]amino]-3-nitrobenzenesulphonate

EC Number:

274-950-1

EC Name:

Sodium 4-[[4-[(4-hydroxy-2-methylphenyl)azo]phenyl]amino]-3-nitrobenzenesulphonate

Cas Number:

70865-20-2

Molecular formula:

C19H16N4O6S.Na

IUPAC Name:

sodium 4-[[4-[(4-hydroxy-2-methylphenyl)azo]phenyl]amino]-3-nitrobenzenesulphonate

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Ibm: GOHI (Himalayan spotted)

Remarks:

SPF-quality guinea pigs delivered by BRL

Sex:

female

Details on test animals and environmental conditions:

ANIMALS
- Strain: Ibm: GOHI; SPF-quality guinea pigs (Himalayan spotted)
- Source: BRL, Biological Research Laboratories Ltd., Wölferstrasse 4, 4414 Füllinsdorf/Switzerland
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
Age at delivery: 5-7 weeks
Age at beginning of pretest/acclimatization period: 6- 8 weeks
- Weight at study initiation: 293 - 498 g
- Housing: individually
- Diet (e.g. ad libitum): pelleted standard Kliba 342, Batch nos. 66/94 (from 16-JAN-1995) and 67/94 (from 27-JAN-1995) guinea pig breeding/maintenance diet (Klingentalmuhle AG,CH-4303 Kaiseraugst), ad libitum.
- Water (e.g. ad libitum): Community tap water ad libitum.
- Acclimation period: One week for the control and test group under test conditions alter health examination.
No acclimatization for the animals of the intradermal pretest and epidermal pretest I.
Four days for the animals used in the epidermal pretest II. Only animals without any visual signs of illness were used for the study.
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 40-80
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 24-Apr-1995 to 03-Jul-1995

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

other: ethanol

Concentration / amount:

Test item: 3% / 0.1 mL / site
Test item / Freund's Complete Adjuvant : 3% / 0.1 mL / site

Day(s)/duration:

one administration on Day 1

Adequacy of induction:

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

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

other: vaselinum album

Concentration / amount:

50%

Day(s)/duration:

Day 8 / 48 hours

Adequacy of induction:

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

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: vaselinum album

Concentration / amount:

5%

Day(s)/duration:

Day 22 / 24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Control group - 10 animals
Test group - 20 animals
Intradermal pretest - 2 animal
Epidermal pretest I - 4 animals
Epidermal pretest II - 4 animals

Details on study design:

RANGE FINDING TESTS:
PRETEST / PERFORMED DURING THE ACCLIMATIZATION PERIOD
The objective of this investigation was to identify a maximally tolerated concentration of the test article suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test article, by the topical route of administration, was identified for the challenge application. The concentrations tested were for the epidermal application the most qualified to assure an optimum technical application procedure and for the intradermal injection the selected concentrations were tested up to 5 % (The Guinea Pig Maximization Test, page 270. Magnusson B.; Kligman A.M., 1969).
The procedure employed for these investigations was as follows:

INTRADERMAL INJECTIONS:
Intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 5, 3 and 1% of the test article in ethanol.
The resulting dermal reactions were assessed 24 hours later. For intradermal induction application a 3% test article dilution was selected.


EPIDERMAL PRETEST I:
Both flanks of each of 4 guinea pigs were clipped and shaved just prior to the application. Thereafter 4 patches of filter paper (2 x 2 cm) were saturated with the test article at A = 25% (this concentration used was found to be the most qualifled to assure an optimum technical application procedure), B = 15%, C = 10% and D = 5% of the test article in vaselinum album and applied to the clipped and shaved flanks. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test article. The dressings were removed alter an exposure period of 24 hours.
Approximately 22 hours after removing of the dressing the application site was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil) to clean the application site from staining produced by the test article, so that possible erythema reactions were clearly visible at that time.
The depilatory was placed on the patch sites and surrounding areas, and left on for 3-5 minutes. It was then thoroughly washed off with a stream of warm, runn-ing water. The animals were then dried with a disposable towel, and returned to their cages. The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema on a numerical basis according to Draize.
For the epidermal induction the test article at 50% in vaselinum album was selected.

EPIDERMAL PRETEST II:
A second epidermal pretest was performed to determine the highest non-irritating concentration used in the challenge procedure. Four additional animals were used and treated in the same conditions as that described previously in the epidermal pretest I. The concentrations of 5%, 3%, 1% and 0.5% in vaselinum album were applied.
For the challenge procedure the test article at 5% in vaselinum album was selected.


MAIN STUDY
INDUCTION
INTRADERMAL INJECTIONS I PERFORMED ON TEST DAY 1
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region as follows:
Test Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test article, diluted to 3 % with ethanol.
3) The test article diluted to 3 % by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Control Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) Ethanol.
3) 1:1 (w/w) mixture of ethanol in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

EPIDERMAL APPLICATIONS/ PERFORMED ON TEST DAY 8
One week after the injections, the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair. A 2 x 4 cm patch of filter paper was saturated with the test article (50% in vaselinum album) and placed over the injection sites of the test animals. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for approximately 48 hours. The epidermal application procedure described ensured intensive contact of the test article.
The guinea-pigs of the control group were treated as described above with vaselinum album only.
Reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.


CHALLENGE / PERFORMED ON TEST DAY 22
The test and control guinea-pigs were challenged two weeks after the epidermal induction application. The test and control guinea-pigs were treated in the same way.
Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea-pig just prior to the application. Two patches (2 x 2 cm) of filter paper were saturated with the highest non-irritating concentration of 5% (left flank) and the vehicle only (vaselinum album, applied to the right flank) using the same method as for the epidermal application. The dressings were left in place for 24 hours.
21 hours alter removal of the dressing the test sites treated with the test article were depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil). The cream was placed on the patch sites for 3-5 minutes and then washed off with a stream of warm running water. When the application sites were clean and any stains from the test article removed the animals were dried with a disposable paper towel and returned to their cages.
24 and 48 hours after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize.

READING OF CHALLENGE REACTIONS
The challenge site was evaluated 24 and 48 hours alter the removal of the patch. The readings were made under artificial fluorescent light (daylight spectrum).

Positive control substance(s):

yes

Results and discussion

Positive control results:

60% of the animals were positive after treatment with a nonirritant test substance concentration of 25% in mineral oil.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5%

No. with + reactions:

8

Total no. in group:

19

Clinical observations:

one animal was found dead on day 11

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5%

No. with + reactions:

9

Total no. in group:

19

Clinical observations:

one animal was found dead on day 11

Remarks on result:

positive indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

10

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

10

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test article applied at a concentration of 5% in vaselinum album is considered to be a sensitizer when used under the described test conditions.

Executive summary:

In order to assess the cutaneous allergenic potential of the test substance, the Maximization-Test in accordance with OECD Guideline No. 406 and the Directive 96154/EEC, B.6 was carried out in 30 (20 test and 10 control) female guinea pigs. In this study eight and nine of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test substance concentration of 5% in vaselinum album after 24 and 48 hours, respectively. No skin reactions were observed in the control group. Therefore the test substance at concentration of 5% in vaselinum album is considered to be a sensitizer when described under the test conditions.