2-[[4,5-dihydro-3-methyl-5-oxo-1-[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]-1H-pyrazol-4-yl]azo]naphthalene-1,5-disulphonic acid, potassium 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:

March 17th, 1992 to April 17th, 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

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:

Study available is over 12 years old

Test material

Test material information

Constituent 1

Reference substance name:

2-[[4,5-dihydro-3-methyl-5-oxo-1-[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]-1H-pyrazol-4-yl]azo]naphthalene-1,5-disulphonic acid, potassium sodium salt

EC Number:

288-950-4

EC Name:

2-[[4,5-dihydro-3-methyl-5-oxo-1-[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]-1H-pyrazol-4-yl]azo]naphthalene-1,5-disulphonic acid, potassium sodium salt

Cas Number:

85940-63-2

Molecular formula:

C22H17N4Na3O13S4

IUPAC Name:

2-[[4,5-dihydro-3-methyl-5-oxo-1-[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]-1H-pyrazol-4-yl]azo]naphthalene-1,5-disulphonic acid, potassium sodium salt

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

No further details specified in the study report.

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 = 292 g (= 100 %)
x min = 266 g (- 8.9 %)
x max= 313 g (+ 7.2 %)
n 15

Randomisation schemes: 347/91
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:

Day 1 / Single application

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:

Day 1 / Single application

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

25%

Day(s)/duration:

Day 22 / 24 hours

Adequacy of challenge:

other: No signs of irritation occurred after epicutaneous administration of different test concentrations up to 25%

No. of animals per dose:

10 animals in the treatment group and 5 animals in the control group were used.

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 sensitising properties
Chronological description of the test procedure indicating the day, at which procedure was carried out:

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-Brilliantgelb GL FWTR in isotonic saline

Day 23
Occlusive bandage removed.

Day 24
Skin examined.

Day 25
Skin examined.
Body weights of test animals determined.

Challenge controls:

Not specified in the study report.

Positive control substance(s):

no

Results and discussion

Positive control results:

No positive control group.

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:

0

Total no. in group:

10

Clinical observations:

Light yellow discolouration

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Light yellow discolouration

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Light yellow discolouration

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Light yellow discolouration

Any other information on results incl. tables

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	312 291 308 295 288	377 370 381 384 370	+ 21 + 27 + 24 + 34 + 28
Treated group:
6 7 8 9 10 11 12 13 14 15	300 276 266 295 272 288 280 303 289 313	373 359 354 378 337 350 346 369 349 396	+ 24 + 30 + 33 + 28 + 24 + 22 + 24 + 22 + 21 + 27

 

Scoring of dermal reactions – individual data

 

Challenge treatment, escort group

Remazol-Brillianygelb GL FWTR 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	19	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-Brillianygelb GL FWTR 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	0 0 X	0 0 X	0 0 X	0 0 X	0 0 X	0 0 X	0 0 X

Time of observation: 72 hours after treatment (Day 25)

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	0 0 X	0 0 X	0 0 X	0 0 X	0 0 X	0 0 X	0 0 X

 

The skin of none of the treated animals (25%) showed a positive reaction during the observation period after the challenge.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, none of ten animals of the treatment group showed a positive skin response after the challenge procedure.
Based on the results of this study Remazol-Brilliantgelb GL FWTR showed no evidence for sensitizing properties.
The substance is not classifiable according to CLP criteria.

Executive summary:

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

 

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

 

Based on the results of this study there is no evidence for sensitizing properties of Remazol-Brilliantgelb GL FWTR.