Benzenesulfonic acid, 2-[2-[5-(aminocarbonyl)-1-ethyl-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-3-pyridinyl]diazenyl]-4-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-, sodium salt (1:2)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 April 1993 to 14 May 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to EU & OECD test guidance in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Details on test material:

See below

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 = 305 g (= 100.0%)
x min = 273 g (-10.5%)
x max - 346 g (+13.4%)
n -15
Randomisation schemes: 614/92
Animal maintenance: in fully air-conditioned rooms in Hakrolon 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 1ibitum
Water: tap water in plastic bottles, ad libitum
Animal identification: fur-marking with KMnO4 and cage numbering

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

physiological saline

Induction 2

Concentration / amount:

25 % in isotonic saline
5 % in isotonic saline
1 % in isotonic saline

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Challenge 2

Concentration / amount:

25 % in isotonic saline
5 % in isotonic saline
1 % in isotonic saline

No. of animals per dose:

Determination of the primary non-irritant concentration: 6
Determination of the tolerance of intradermal injections: 3
Escort group: 5
Control group: 5
Treatment group: 10

Details on study design:

The following vehicles were used:
Freund's Complete Adjuvant (Behringwerke AG, Marburg)
isotonic saline (sterile, pyrogen-free; Fresenius AG, Bad Homburg)

Freund's Complete Adjuvant was mixed immediately before use with an equal volume of physiological saline. This 50 % adjuvant solution was administered to the animals by intradermal injection.

For the dermal and intradermal treatments, Reaktiv-Gelb F-66 923 FW was applied in isotonic saline [percentages w/v].

For the intradermal injections of the test substance in 50 % Freund's adjuvant, Reaktiv-Gelb F-66 923 FW was diluted with isotonic saline and then mixed with an equal volume of Freund's Original Adjuvant [percentages w/v].

The concentrations for the maximization test cannot be standardized. Suitable concentrations are established in preliminary tests. The selected concentration of the test substance depends on the individual phases of the study.

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 pre-parations was administered twice by interdermal 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.

Challenge controls:

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 % Reaktiv-Gelb F-66 923 FW in isotonic saline

Positive control substance(s):

no

Study design: in vivo (LLNA)

Vehicle:

other: not applicable

Results and discussion

Positive control results:

Not applicable.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Erythema reactions

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Erythema reactions.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Oedema reactions

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Oedema reactions.

Results 3

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Erythema reactions

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Erythema reactions.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Oedema reactions

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Oedema reactions.

Results 5

Reading:

1st reading

Hours after challenge:

72

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Erythema reactions

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 72.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Erythema reactions.

Results 6

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Oedema reactions

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Oedema reactions.

Results 7

Reading:

1st reading

Hours after challenge:

72

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Erythema reactions

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Erythema reactions.

Results 8

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Oedema reactions

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Oedema reactions.

In vivo (LLNA)

Results

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Not applicable

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 i rritation determined in pre!imi nary tests. For this reason, the five animals in the escort group which had been treated with Freund's Adjuvant were treated with 25%Reaktiv-Gelb F-66 923 FW in isotonic saline. As no reac­tions were observed in these animals, a concentration of 25%Reaktiv-Gelb F-66 923 FW in isotonic saline was chosen for the challenge at day 22.

 

Tolerance of intradermal injections

 

The intradermal injections with the 0.2%preparation caused no irritations.

Very slight oedema as well as barely perceptible erythema occurred after appli­cation of the 1%preparation.

The intradermal injections with the 5%preparation revealed very slight up to

well-defined erythema as well as very slight to slight oedema.

Additonally encrusted injection sites were observed after application of the 5 %

preperation.

Furthermore the intrademal injections with 0.2 %, 1 % and 5 % preparations were discoloured light yellow.

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

 

Main test for sensitizing 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 wel 1 as indurated and encrusted skin. The intradermal injections with the test substance in the vehicle caused very slight up to slight oedema as well as well-defined erythema. The intrader­mal applications of the vehicle caused no signs of irritation. Additionally the application sites treated with the test substance in Freund's Adjuvant and in the vehicle showed yellowish discolourations.

Due to these strong irritation reaction of the skin, 10% sodium dodecylsulfate was not applied at day 7.

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 light yellow discoloured 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 and in the treated group.

 

Treated area: left flank

Time of observation: 48 hours after treatment
Scoring of dermal reactions
Control animals	1	2	3	4	5
Erythema	0	0	0	0	0
Oedema	0	0	0	0	0
Yellowish discoloured	X	X	X	X	X
Treated animals	6	7	8	9	10	11	12	13	14	15
Erythema	0	0	0	0	0	0	0	0	0	0
Oedema	0	0	0	0	0	0	0	0	0	0
Yellowish discoloured	X	X	X	X	X	X	X	X	X	X
Time of observation: 72 hours after treatment
Scoring of dermal reactions
Control animals	1	2	3	4	5
Erythema	0	0	0	0	0
Oedema	0	0	0	0	0
Yellowish discoloured	X	X	X	X	X
Treated animals	6	7	8	9	10	11	12	13	14	15
Erythema	0	0	0	0	0	0	0	0	0	0
Oedema	0	0	0	0	0	0	0	0	0	0
Yellowish discoloured	X	X	X	X	X	X	X	X	X	X

None of the treated animals showed a positive reaction during the observation period after the challenge

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

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 Reaktiv-Gelb F-66 923 FW showed no evidence for sensitising properties.

Executive summary:

The objective of the test procedure described here is to determine the potential sensitising properties of a substance. 

This study was conducted in compliance with EC-Guideline B.6. Acute Toxicity Sensitisation of the Skin of the Directive 92/69/EEC and OECD-Guideline for testing of chemicals, 406 "Skin Sensitisation". Study conducted in compliance with GLP.

 

Testing for sensitising properties of Reaktiv-Gelb F-66 923 FW was performed in female Guinea pigs according to the method of MAGNUSSON & KLIGMAN.

 

Intradermal induction was performed using 5.0 % Reaktiv-Gelb F-66 923 FW in isotonic saline. Dermal induction and challenge treatment were carried out with 25 % Reaktiv-Gelb F-66 923 FW in isotonic saline.

 

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% Reaktiv-Gelb F-66 923 FW in isotonic saline. As no reactions were observed in these animals, a concentration of 25% Reaktiv-Gelb F-66 923 FW in isotonic saline was chosen for the challenge.

 

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

 

No signs of irritation were observed 24 and 48 hours after removal of the occlusive bandage in the control group and in the treated group.

 

Based on the results of this study there is no evidence for sensitising properties of Reaktiv-Gelb F-66 923 FW.