Sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 19, 1994 to October 13, 1994

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Available study information is over 12 years old.

Test material

Test material information

Constituent 1

Reference substance name:

Sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate

EC Number:

266-867-4

EC Name:

Sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate

Cas Number:

67674-28-6

Molecular formula:

C26H25N5O5S.Na

IUPAC Name:

sodium m-[[4-[[p-[bis(2-hydroxyethyl)amino]phenyl]azo]-1-naphthyl]azo]benzenesulphonate

Test material form:

solid: particulate/powder

Details on test material:

Acid Red 299

In vivo test system

Test animals

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

The albino guinea pig is the recommended species for skin sensitisation studies.
Animal strain: Pirbright White Strain (Tif: DHP)
Breeder: CIBA-GEIGY Limited, Animal Production 4332 Stein, Switzerland
Date of acclimatisation: September 14, 1994

Group Size and Husbandry
The test was performed on 5 male and 5 female guinea pigs in the test group and 5 female guinea pigs in the control group, initially weighing between 358 to 425 g.
The animals were housed individually in Macrolon cages (Type 3), assigned to the different groups by means of random numbers generated by the random number generator, identified by individual ear tags, kept at a constant room temperature of 22 ± 3 °C, at a relative humidity of 30 to 70% and a 12 hours light cycle day.
The animals received ad libitum standard guinea pig pellets - NAFAG No. 845, Gossau SG and fresh water.
All batches of the diet are assayed for nutritive ingredients and contamination level by the manufacturer.
The drinking water quality fulfilled the critical parameters in the specifications of the 11 Schweizerisches Lebensmittelbuch 11 (Edition 1972). The results of the routine chemical examination of water at source (Grundwasserfassung Stein) as conducted periodically by the water authority (Baudepartement des Kantons Aargau, Abteilung Gewaesserschutz) are available to CIBA-GEIGY Limited, as well as the results of in house chemical analysis by the analytical laboratories of the Pharmaceutical Division, CIBA-GEIGY Limited.

Sensitivity of strain
The sensitivity of the strain is checked once or twice a year with a known mild to moderate sensitiser, such as mercaptobenzothiazole, hexyl cinnamic aldehyde or potassiumdichromate.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

5%

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:

intradermal

Vehicle:

physiological saline

Concentration / amount:

5% in Freund's adjuvant/saline mixture 1:1

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 3

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

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:

physiological saline

Concentration / amount:

10%

Day(s)/duration:

day 22/24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

5 male and 5 female guinea pigs in the test group and 5 female guinea pigs in the control group

Details on study design:

Pretests
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 of test article has been used for intradermal injection:
5% in physiological saline (w/v).
Since 5% FAT 21071/G 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 following concentrations of FAT 21071/G have been examined on separate animals for the determination of the maximum subirritant concentration:
10, 20, 30, and 50% in physiological saline.
50% was the highest possible concentration of the test article in physiological saline.
Reactions were observed with 20, 30, and 50% FAT 21071/G in physiological saline.

Test procedure
DAY 0: INDUCTION, intradermal injections
Three pairs of intradermal injections (0.1 ml per injection) were made simultaneously into the left and right side of the shaved neck of the test and control group animals.
Test group:
-adjuvant/saline mixture 1:1 (v/v)
-5% FAT 21071/G in physiological saline (w/v)
-5% FAT 21071/G 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

DAY 8: INDUCTION, epidermal application
In the test group FAT 21071/G was incorporated in physiological saline and applied on a filte rpaper patch to the neck of the animals (patch 2 x 4 cm; approx. 0.4 g per patch; occluded administration for 48 hours).
The control group was treated with the vehicle only.
Test group:
- 50% FAT 21071/G in physiological saline
Control group:
- physiological saline only

DAY 21: Challenge
The test and control group animals were tested on one flank with FAT 21071/G in physiological saline and on the other flank with the vehicle alone (patch 2 x 2 cm; approx. 0.2 g per patch; occluded administration for 24 hours).
Test and control group:
- 10% FAT 21071/G in physiological saline
- physiological saline only

Observations and records
Induction reactions
After removal of the dressing on day 10, irritation of the epidermal application site was observed in 10/10 test group animals.

Challenge reactions
Twenty four and forty eight hours after removing the dressings, the challenge reactions were graded according to the Draize scoring scale.

General
The body weight was recorded at start and end of the test.

Challenge controls:

The vehicle alone was applied to one flank of each animal.

Positive control substance(s):

yes

Remarks:

2-Mercaptobenzothiazole puriss.

Results and discussion

Positive control results:

Number of positive animals per group after occlusive epidermal application
Control group:
after 24 hours after 48 hours
vehicle control 0/10 0/10
test article 0/10 0/10

Test group:
after 24 hours after 48 hours
vehicle control 0/20 0/20
test article 20/20 20/20

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

6

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

30%

No. with + reactions:

20

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

30%

No. with + reactions:

20

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Results 7

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

10%

No. with + reactions:

0

Total no. in group:

5

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

10%

No. with + reactions:

0

Total no. in group:

5

Results 9

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

vehicle

No. with + reactions:

0

Total no. in group:

5

Results 10

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

vehicle

No. with + reactions:

0

Total no. in group:

5

Any other information on results incl. tables

Number of positive animals per group after occlusive epidermal application

Control group:
	After 24 hours	After 48 hours
Vehicle control	0/5	0/5
Test article	0/5	0/52
Test group:
	After 24 hours	After 48 hours
Vehicle control	0/10	0/10
Test article	7/10	6/10

 

Challenge reactions after epidermal application

(CONTROL GROUP)

DRAIZE Score 24 hours after removal of the dressing

Vehicle control
Female animals	151	152	153	154	155
Erythema score: Edema score	0 0	0 0	0 0	0 0	0 0
Test article control
Female animals	151	152	153	154	155
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0

DRAIZE Score 48 hours after removal of the dressing

Vehicle control
Female animals	151	152	153	154	155
Erythema score: Edema score	0 0	0 0	0 0	0 0	0 0
Test article control
Female animals	151	152	153	154	155
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0

 

Challenge reactions after epidermal application

(TEST GROUP)

DRAIZE Score 24 hours after removal of the dressing

Vehicle control
Male animals	146	147	148	149	150
Erythema score: Edema score	0 0	0 0	0 0	0 0	0 0
Female animals	156	157	158	159	160
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0
Test article control
Male animals	146	147	148	149	150
Erythema score: Edema score:	1 1	1 0	1 1	1 0	1 1
Female animals:	156	157	158	159	160
Erythema score: Edema score:	0 0	0 0	1 0	1 0	0 0

DRAIZE Score 48 hours after removal of the dressing

Vehicle control
Male animals	146	147	148	149	150
Erythema score: Edema score	0 0	0 0	0 0	0 0	0 0
Female animals	156	157	158	159	160
Erythema score: Edema score:	0 0	0 0	0 0	0 0	0 0
Test article control
Male animals	146	147	148	149	150
Erythema score: Edema score:	1 1	1 0	1 1	0 0	1 1
Female animals:	156	157	158	159	160
Erythema score: Edema score:	0 0	0 0	1 1	1 1	0 0

 

Evaluation of the primary skin irritation potential

Procedure: On each animal 2 concentrations of FAT 21071/G were applied simultaneously on the left and right flank. A naïve skin site served as control.

At least 7 days before application of FAT 21071/G two 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.

 

	Score 24 hours		Score 48 hours
	After removing the dressing
Concentrations of FAT 21071/G in physiological saline
Animal No./ Sex	30% er/ed	50% er/ed	30% er/ed	50% er/ed
1 male 2 female	0/0 1/1	½ 1/1	0/0 0/0	1/2 1/1
Animal No./ Sex	10% er/ed	20% er/ed	10% er/ed	20% er/ed
3 male 4 female	0/0 0/0	1/0 1/0	0/0 0/0	1/0 1/0

er = erythema, ed = edema

 

Individual animal bodyweights in g

Test group males

Animal No.	Weight
	At start	At end
146 147 148 149 150	425 418 419 377 407	574 626 517 507 540
Mean Std. Dev.	409 19.1	553 48.3

Test group Females

Animal No.	Weight
	At start	At end
156 157 158 159 160	378 358 367 377 406	503 449 440 465 529
Mean Std. Dev.	377 18.0	477 37.7

Control group females

Animal No.	Weight
	At start	At end
151 152 153 154 155	380 401 364 369 409	524 563 489 518 539
Mean Std. Dev.	385 19.7	527 27.3

 

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the experimental conditions employed, 70% and 60% of the animals of the test group showed skin reactions 24 and 48 hours after removing the dressings, respectively.
According to the maximisation grading of Magnusson and Kligman Acid Red 299 showed a moderate to strong grade of skin-sensitising (contact allergenic) potential in albino guinea pigs.

Executive summary:

Body weights were not affected by treatment.

Under the experimental conditions employed, 70% and 60% of the animals of the test group showed skin reactions 24 and 48 hours after removing the dressings, respectively.

 

Acid Red 299, therefore, classified as a moderate to strong sensitiser in albino guinea pigs according to the grading of Magnusson and Kligman.