4-[[2-[[2,5-dimethoxy-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1,3-dioxobutyl]amino]-5-methoxy-2-methylbenzenesulphonic acid, 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:

June 30th, 1992 to August 07th, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

OECD-Guideline for testing of chemicals, 406 "Skin Sensitization", Adopted 12 May 1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

EG-Guideline B.6. Acute Toxicity Sensitization of the Skin of the Directive 84/449/EWG:
Commission Directive of 25 April 1984 adapting to technical progress for the sixth time Council Directive 67/548/EWG on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Available study data is over 12 years old.

Test material

Test material information

Constituent 1

Reference substance name:

4-[[2-[[2,5-dimethoxy-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1,3-dioxobutyl]amino]-5-methoxy-2-methylbenzenesulphonic acid, sodium salt

EC Number:

303-161-8

EC Name:

4-[[2-[[2,5-dimethoxy-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1,3-dioxobutyl]amino]-5-methoxy-2-methylbenzenesulphonic acid, sodium salt

Cas Number:

94158-87-9

Molecular formula:

C22H(27-x)N3NaxO14S3 x<=2 C22H27N3O14S3.xNa

IUPAC Name:

4-[[2-[[2,5-dimethoxy-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1,3-dioxobutyl]amino]-5-methoxy-2-methylbenzenesulphonic acid, sodium salt

Test material form:

solid: particulate/powder

Details on test material:

Reactive Yellow 160

In vivo test system

Test animals

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

female

Details on test animals and environmental conditions:

Test species: Pirbright-White guinea pig
Sex: female
Strain: Hoe: DHPK (SPFLac)
Origin: HOECHST AG, Kastengrund, SPF breeding colony
Body weight at start of study:
X = 307 g (= 100.0 %)
x min = 277 g (- 9.8 %)
x max = 338 g (+ 10.1 %)
n 15

Randomisation schemes: 383/92
Animal maintenance: in fully air-conditioned rooms in Makrolon cages (Type 4) on soft wood granulate, in groups of 5 animals
Ambient temperature: 22 ± 3 °C
Rel . atmospheric humidity: 55 ± 20 %
Lighting time: 12 hours daily
Acclimatisation: at least 5 days
Diet: Altromin 3112 for guinea pigs and rabbits, ad libitum
Water: tap water in plastic bottles, ad libitum
Animal identification: fur-marking with KMn04 and cage numbering

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:

other: 50% Freund's adjuvant

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 3

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

25%

Day(s)/duration:

day 8/48 hours

Adequacy of induction:

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

Challenge

Challenge 1

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

25%

Day(s)/duration:

day 22/24 hours

Adequacy of challenge:

highest non-irritant concentration

Challenge 2

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

25%

Day(s)/duration:

day 29/24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Treatment group: 10
Control group: 5

Details on study design:

Determination of the primary non-irritant concentration
In a dermal-occlusive test for primary skin irritation, each of the following test concentrations was applied to the left flank of two guinea pigs:
25% in isotonic saline
5% in isotonic saline
1% in isotonic saline
The hair on the left flank of the animals was removed mechanically. 0.5 ml of the test substance preparation was applied to a 2 x 2 cm cellulose patch, which was then fixed to the left flank and covered occlusively for 24 hours with a bandage and film. 24 hours after removal of the patches, the treated skin areas were examined for erythema and oedema.

Determining of the tolerance of intradermal injections
To determine the tolerance of intradermal injections, each of the following preparations was administered twice by intradermal injection to 3 guinea pigs. The injection sites (sites 1, 2 and 3) were all within a dorsal area measuring 2 x 4 cm in the vicinity of the shoulder.
site appl. vol.in ml conc. in % vehicle
1 2 X 0.1 5.0 isotonic saline
2 2 X 0.1 1.0 isotonic saline
3 2 X 0.1 0.2 isotonic saline

The injection sites (site 1, 2 and 3) were all within a dorsal area measuring 2 x 4 cm in the vicinity of the animals' shoulder.

Main test for sensitisinq properties
Chronological description of the test procedure indicating the day, at which procedure was carried out, on the left margin of the page:
Day 0
The body weights of animals were determined.
The guinea pigs were shaved mechanically over a dorsal area of 4 x 6 cm in the vicinity of the shoulders.

Day 1
Intradermal induction treatment
Two intradermal injections per animal of the following preparations.
The injection sites (site 1, 2 and 3) were all within a dorsal area of 2 x 4 cm. The injection sites were left uncovered.
Treated group:
Site 1: 2 X 0.1 ml 50% Freund's Adjuvant
Site 2: 2 X 0.1 ml 5% solution of test substance in isotonic saline
Site 3: 2 X 0.1 ml 5 % solution of test substance in 50% Freund's adjuvant

Control and escort groups:
Site 1: 2 X 0.1 ml 50% Freund's Adjuvant
Site 2: 2 X 0.1 ml isotonic saline
Site 3: 2 X 0.1 ml 50% Freund's Adjuvant

Days 1-7
The application area was examined for local tolerance. Any systemic toxic effects were recorded.

Day 8
Dermal induction treatment
0.5 ml of the test substance preparation or the vehicle was applied to a 2 x 4 cm cellulose patch. This patch covered the area where the intradermal injection had been placed. The application area was then kept for 48 hours under an occlusive bandage with an impermeable film and an elastic bandage.
Treated group: 25% test substance in isotonic saline
Control and escort group: isotonic saline

Day 10
Occlusive bandage removed.
Irritant effects recorded.

Days 11-21
No treatment of control or treated group.
Test animals kept under observation.

Days 15-18
Challenge treatment of escort group, carried out in same way as that of control and treated groups (see days 22 - 25).

Day 22
Dermal challenge treatment
One area of approx. 5 x 5 cm on the left flank was shaved mechanically.
0.5 ml of the test substance preparation was applied to a 2 x 2 cm cellulose patch. The application area was then kept for 24 hours under an occlusive bandage with an impermeable film and an elastic bandage.
Treated and control groups (left flank):
25% Remazol-Brillantgelb 4GL in isotonic saline

Day 23
Occlusive bandage removed.

Day 24
Skin examined.

Day 25
Skin examined.

Day 29
Repeated dermal challenge treatment
Treated and control groups (right flank):
25% Remazol-Brillantgelb 4GL in isotonic saline
(Handling analogous to the first dermal challenge treatment)

Evaluation
Erythema and oedema are major clinical indicators of an allergic reaction. The decisive criterion for evaluation of the sensitising properties of a test substance is the number of sensitised test animals, not the intensity of the dermal reaction.
The substance is considered to be sensitising if 30% of the animals in the treated group definitely show a positive reaction.

Positive control substance(s):

yes

Results and discussion

Positive control results:

valid

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

light yellow discoloration

Results 2

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

light yellow discoloration

Results 3

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

light yellow discoloration

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

light yellow discoloration & dry, rough skin

Results 5

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

light yellow discoloration

Results 6

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

light yellow discoloration & encrusted

Results 7

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

light yellow discoloration

Results 8

Key result

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

light yellow discoloration, dry, rough & encrusted skin

Any other information on results incl. tables

Determination of the primary non-irritant concentration

No signs of irritation occurred after application of the different test concentrations.

Treatment of the animals with Freund's Adjuvant can lower the threshold value for primary irritation determined in preliminary tests. For this reason, the five animals in the escort group which had been treated with Freund's Adjuvant were treated with 25 % Remazol-Brillantgelb 4GL in isotonic saline. As no reactions were observed in these animals, a concentration of 25% Remazol-Brillantgelb 4GL in isotonic saline was chosen for the challenge at day 22.

 

Tolerance of intradermal injections

The intradermal injections with the 0.2 % and 1.0 % preparations caused very slight erythema and barely perceptible oedema. Very slight to well-defined erythema and very slight to slight oedema occurred after application of the 5 % preparation.

Based on this preliminary test, a 5 % preparation was selected for the intradermal injections in the main test.

 

Main test for sensitising properties

Body weight gains and clinical signs

The treated animals showed no clinical signs of intoxication throughout the study.

The intradermal injections with Freund's Adjuvant (with and without test substance) caused severe erythema and oedema as well as indurated, encrusted and scabbed skin. The application sites treated with the test substance in the vehicle exhibited slight erythema and oedema. Intradermal applications of the vehicle caused no signs of irritation. Additionally the application sites treated with the test substance in Freund's Adjuvant showed yellowish discolorations.

After the removal of the patch at day 10, erythema and oedema, scabbed and encrusted skin as well as necrosis and open wounds were observed at the sites previously treated with Freund's Adjuvant. The injection sites treated with the test substance in the vehicle and the vehicle alone showed no signs of irritation.

Additionally yellow discolored skin was noted in the animals of the treatment group.

The body weight gains of the treated animals were not impaired.

 

Challenge treatment

No signs of irritation were observed 24 and 48 hours after removal of the occlusive bandage in the control group. 48 hours after removal of the occlusive bandage three animals of the treated group showed very slight erythema.

After the repeated dermal challenge treatment the animals of the control group showed no signs of irritation. 48 hours after removal of the occlusive bandage very slight up to moderate erythema were noted at the skin of seven animals of the treated group. Additionally, very slight oedema as well as dry, rough and encrusted skin were observed.

Body weight gains

Animal No.	Body weight at start of study (g)	Body weight at end of study (g)	Increase (%)
Control group:
1 2 3 4 5	288 291 310 322 319	365 390 445 437 426	+27 +34 +44 +36 +34
Treated group:
6 7 8 9 10	289 306 319 277 303	344 402 391 347 374	+19 +31 +23 +25 +23
11 12 13 14 15	294 338 305 326 311	375 404 385 419 355	+28 +20 +26 +29 +14

 

Scoring of dermal reactions – individual data

 

Challenge treatment, escort group

Remazol-Brillantgelb 4GL 25% in isotonic saline (day 15)

Treated area: left flank

Scoring of dermal reactions

	Animal No.:	16	17	18	19	20
48 hours p.a.	Erythema Oedema Light yellow discoloured	0 0 X	0 0 X	0 0 X	0 0 X	0 0 X
	Animal No.:	16	17	18	18	20
72 hours p.a.	Erythema Oedema Light yellow discoloured	0 0 X	0 0 X	0 0 X	0 0 X	0 0 X

 

Challenge treatment: Remazol-Brillantgelb 4GL 25% in isotonic saline (day 22)

Treated area: left flank

 

Time of observation: 48 hours after treatment (day 24)

Scoring of dermal reactions

Control animals	1	2	3	4	5
Erythema Oedema Light yellow discol.	0 0 X	0 0 X	0 0 X	0 0 X	0 0 X
Treated animals	6	7	8	9	10	11	12	13	14	15
Erythema Oedema Light yellow discol.	0 0 X	0 0 X	0 0 X	1 0 X	0 0 X	0 0 X	0 0 X	1 0 X	0 0 X	0 0 X

 

Time of observation: 72 hours after treatment (day 24)

Scoring of dermal reactions

Control animals	1	2	3	4	5
Erythema Oedema Light yellow discol.	0 0 X	0 0 X	0 0 X	0 0 X	0 0 X
Treated animals	6	7	8	9	10	11	12	13	14	15
Erythema Oedema Light yellow discol. Dry, rough	0 0 X	0 0 X	0 0 X	1 0 X X	0 0 X	0 0 X	1 0 X	1 0 X X	0 0 X	0 0 X

 

Repeated challenge treatment: Remazol-Brillantgelb 4GL 25% in isotonic saline (day 29)

Treated area: left flank

 

Time of observation: 48 hours after treatment (day 31)

Scoring of dermal reactions

Control animals	1	2	3	4	5
Erythema Oedema Light yellow discol.	0 0 X	0 0 X	0 0 X	0 0 X	0 0 X
Treated animals	6	7	8	9	10	11	12	13	14	15
Erythema Oedema Light yellow discol. Encrusted	0 0 X	1 0 X	0 0 X	1 0 X	0 0 X	1 0 X	2 0 X	2 1 X X	0 0 X	1 0 X

 

Time of observation: 72 hours after treatment (day 32)

Scoring of dermal reactions

Control animals	1	2	3	4	5
Erythema Oedema Light yellow discol.	0 0 X	0 0 X	0 0 X	0 0 X	0 0 X
Treated animals	6	7	8	9	10	11	12	13	14	15
Erythema Oedema Light yellow discol. Dry, rough Encrusted	0 0 X	1 0 X X	0 0 X	1 0 X X	0 0 X	1 0 X	2 1 X X	3 1 X X X	1 0 X	2 0 X X

 

The skin of seven treated animals showed a positive reaction during the observation period after the challenge.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the conditions of the study, seven of ten animals of the treatment group showed a positive skin response after the challenge procedure.
Thus, the percentage of animals reacting positive is above the threshold of 30%.
Based on the results of this study Remazol-Brillantgelb 4GL may cause sensitisation by skin contact.

Executive summary:

Testing for sensitizing properties of Remazol-Brillantgelb 4GL was performed in female Guinea pigs according to the method of MAGNUSSON & KLIGMAN .

Intradermal induction was performed using 5.0 % Remazol -Brillantgelb 4GL in isotonic saline. Dermal induction and challenge treatment were carried out with 25% Remazol-Brillantgelb 4GL in isotonic saline.

Based on the results of this study Remazol-Brillantgelb 4GL may cause sensitisation by skin contact.