REAKTIV-GELB F-68 072 FW

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 1993 to 04 June 1993

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:

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

Justification for non-LLNA method:

Study was already available

Test material

Test material information

Constituent 1

Reference substance name:

lithium sodium 2-amino-4-{[4-(cyanoazanidyl)-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl]amino}-5-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)benzene-1-sulfonate

EC Number:

413-090-5

Cas Number:

189574-45-6

Molecular formula:

Hill formula: C24H18LixN10NayO12S4

IUPAC Name:

lithium sodium 2-amino-4-{[4-(cyanoazanidyl)-6-[(3-sulfonatophenyl)amino]-1,3,5-triazin-2-yl]amino}-5-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)benzene-1-sulfonate

Test material form:

solid: particulate/powder

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 = 335 g (= 100 %)
x min = 314 g (- 2.3 %)
x max = 348 g (+3.8 %)
n =15
Randomisation schemes: 411/92
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 % solution of test substance in istonic saline / 2 x 0.1 ml
5 % solution of test substance in 50% Freund's adjuvant / 2 x 0.1 ml

Day(s)/duration:

Day 1

Adequacy of induction:

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

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Concentration / amount:

25 % test substance in isotonic saline / 0.5 mL

Day(s)/duration:

Day 8 / 48 h

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% / 0.5 mL

Day(s)/duration:

Day 22 / 24 h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Group No. of animals
Determination of the primary non-irritant concentration 6
Determination of the tolerance of intradermal injection 3
Escort group 5
Control group 5
Treatment group 10

Details on study design:

10 animals in the treatment group and 5 animals in the control group were used. Based on experience these animal numbers are sufficient for the determination of the sensitising properties of test substances.

In case of a questionable result additional animals will be tested to give a total of 20 test and 10 control animals.

Preparation of test concentrations
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 treatments, Reaktiv-Gelb F-68 072 FW was applied in isotonic saline [percentages w/v]. For the intradermal injections, Reaktiv-Gelb F-68 072 FW was diluted with isotonic saline [percentages w/v].

For the intradermal injections of the test substance in 50 % Freund's adjuvant, Reaktiv-Gelb F-68 072 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 maximisation 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 flanks 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 2x4 cm in the vicinity of the shoulder.

Main test for sensitizing 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 (see prior page). The injection sites were left uncovered.

Treated Group:

Site 1: 2 x 0.1 ml 50 % Freund's Adjuvant 2 x 0.1 ml
Site 2: 2 x 0.1 ml 5 % solution of test substance in istonic saline
Site 3: 2 x 0.1 ml 5% solution of test substance in 50% Freunds adjuvant

Control and escort groups:

Site 1: 2 x 0.1 ml 50% Freunds Adjuvant
Site 2: 2 x 0.1 ml isotonic saline
Site 3: 2 x 0.1 ml 50% 50% Freunds Adjuvant

Days 1-6
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 % Reaktiv-Gelb F-68 072 FW in isotonic saline.

Day 23
Occlusive bandage removed.

Day 24
Skin examined.

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

Challenge controls:

patch with Physiological saline in treatment and control groups

Positive control substance(s):

yes

Remarks:

bi-annual testing

Results and discussion

Positive control results:

valid - the bi-annually conducted positive control showed sufficient skin sensitisation

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Results 2

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

25

No. with + reactions:

0

Total no. in group:

5

Results 3

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

48

Group:

positive control

Remarks on result:

positive indication of skin sensitisation

Results 6

Reading:

2nd reading

Hours after challenge:

72

Group:

positive control

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

Challenge treatment: Reaktiv-Gelb F-68 072 FW 25% in isotonic salin

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
Skin discoloured orange	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
Skin discoloured orange	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
Skin discoloured orange	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
Skin discoloured orange	X	X	X	X	X	X	X	X	X	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.

Thus, the percentage of animals reacting positive is below the threshold of 30%.

Based on the results of this study Reaktiv-Gelb F-68 072 FW showed no evidence for sensitising properties.

Executive summary:

Chemical substances may produce an immunological reaction (sensitisation) after single or repeated dermal application. Further application after the elapse of a certain period may cause allergic reactions. The objective of the test procedure described here is to determine the potential sensitizing properties of a substance.

 

This study was conducted in compliance with EC-Guideline B.6. Acute Toxicity Sensitization of the Skinof the Directive 92/69/EWG and OECD-Guideline for testing of chemicals, 406 "Skin Sensitization". This study was conducted in compliance with the Principles of Good Laboratory Practice(GLP).

 

Testing for sensitising properties of Reaktiv-Gelb F-68072FW was performed in female Guinea pigs according to the method of MAGNUSSON & KLIGMAN.

 

Intradermal induction was performed using 5% Reaktiv-Gelb F-68072FW in isotonic saline. Dermal induction and challenge treatment were carried out with 25% Reaktiv-Gelb F-68072FW in isotonic saline.

 

Based on the results of this study Reaktiv-Gelb F-68 072 FW showed no evidence of sensitising properties. The substance does not trigger classification and labelling for sensitisation endpoint.