Benzoic acid, 2-hydroxy-5-[[4-[[4-[[8-hydroxy-7-[[4-[(8-hydroxy-3,6-disulfo-1-naphthalenyl)azo]-2-methoxy-5-methylphenyl]azo]-3,6-disulfo-1-naphthalenyl]amino]-6-(phenylamino)-1,3,5-triazin-2-yl]amino]phenyl]azo]-, pentasodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 22 to December 16, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

1992

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Guinea pig maximisation test was available.

Test material

Test material information

Constituent 1

Reference substance name:

Direct Green 026

IUPAC Name:

Direct Green 026

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Pirbright white (Tif: DHP)

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 300 - 393 g
- Housing: individually
- Diet: ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Photoperiod: 12 h light

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

physiological saline

Remarks:

and in adjuvant/saline mixture

Concentration / amount:

5 %, 0.1 ml

Day(s)/duration:

day 0

Adequacy of induction:

other: well-tolerated

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

50 %, 0.4 g

Day(s)/duration:

day 8

Adequacy of induction:

highest technically applicable concentration used

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

petrolatum

Concentration / amount:

30 %, 0.2 g

Day(s)/duration:

day 21

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10/sex

Details on study design:

RANGE FINDING TESTS:
Intradermal Induction
The concentration for the intradermal injections was selected on account of the solubility of the test article in standard vehicles and its local and systemic tolerability in a pretest.
The following concentration was used for intradermal injection: 5 % in physiological saline (w/v).
Since 5 % test substance in physiological saline could be injected and was well tolerated, this concentration was used for the intradermal induction.

Epidermal applications (induction and challenge)
The concentrations for the epidermal applications were selected on account of the primary irritation potential of the test article. The concentrations of 30 % and 50 % in vaseline were examined on separate animals for the determination of the maximum subirritant concentration.
50 % in vaseline was the highest applicable concentration of the test article. Erythema reactions were observed with 50 % test substance in vaseline.

MAIN STUDY
A. INDUCTION EXPOSURE - INTRADERMAL
- No. of exposures: 1
- Exposure period: day 0 (intradermal)
- Test group:
adjuvant/saline mixture 1:1 (v/v)
5% in physiological saline (w/v)
5% in the adjuvant/saline mixture (w/v)
- Control group:
adjuvant/saline mixture 1:1 (v/v)
adjuvant/saline mixture 1:1 (v/v)
physiological saline
- Site: left and right side of the shaved neck

A. INDUCTION EXPOSURE - EPICUTANEOUS
- No. of exposures: 1
- Application: day 8, with filterpaper patch (2 × 4 cm; occluded)
- Exposure period: 48 h
- Pretreatment: the application site was pretreated with 10 % sodium-laurylsulfate (open application) 24 hours prior to the epidermal induction application.
- Test group: 50 % in vaseline
- Control group: vaseline only
- Site: neck

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: day 21
- Exposure period: 24 h
- Test group: 30 % in vaseline
- Control group: vaseline only
- Site: flank
- Concentrations: 30 %
- Evaluation: 24 and 48 h after removing the dressing

Positive control substance(s):

yes

Remarks:

with 2-mercaptobenothiazole, August 1993

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5 %

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

very slight erythema

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5 %

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no 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

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

30%

No. with + reactions:

9

Total no. in group:

10

Remarks on result:

other: positive control mercaptobenzothiazole was tested once a year, not concurrent to the test.

Remarks:

The following concentrations of the reference compound and vehicles were used: Intradermal induction Concentration of compound: 5% Vehicle: Oleum arachidis Epidermal induction Concentration of compound: 50% Vehicle: vaseline Epidermal challenge Concentration of compound: 30% Vehicle: vaseline

Any other information on results incl. tables

Evaluation of the primary skin irritation potential

Procedure: on each animal, 2 concentrations of test substance were applied simultaneously on the left and right flank. A naive skin site served as control (not reported). 7 days before application of test substance, 2 pairs of intradermal injections of an adjuvant/saline mixture 1:1 (v/v; 0.1 ml per injection) were made simultaneously into the shaved neck of the guinea pigs.

animal/sex	24 h				48 h
	30 %	50 %	30 %	50 %	30 %	50 %	30 %	50 %
	er.	oed.	er.	oed.	er.	oed.	er.	oed.
1 male	0	0	0	0	0	0	0	0
1 female	0	0	1	0	0	0	0	0

er. = erythema

oed. = oedema

Applicant's summary and conclusion

Interpretation of results:

other: not classified according to the CLP Regulation (EC 1272/2008)

Conclusions:

Non skin sensitising in a guinea pig maximisation test.

Executive summary:

Method

Guinea pig maximisation test using 20 animals (10/sex) in test group and 10 animals (5/sex) in control group. A pretest was conducted to choose test substance concentration for intradermal and epidermal applications.

Intradermal induction was done on day 0 using a 5 % solution of test substance in physiological saline and adjuvant/saline mixture.

Epidermal induction was done on day 8, applying test substance at 50 % in vaseline for 48 h. Challenge was done on day 21, applying test substance at 30 % in vaseline for 24 h. Readings were done 24 and 48 h after removal of the dressings.

Results

5 % test substance in physiological saline was well tolerated; 50 % in vaseline was the highest applicable concentration, causing erythema reactions.

Upon challenge on day 21, 2/20 positive responses were reported in test group after 24 h, which were no longer evident after 48 h..