4,4'-[(6-chloro-1,3,5-triazine-2,4-diyl)diimino]bis[5-hydroxy-6-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonic] 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:

12. October to 27 November 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:

1993

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

84/449/EWG, B.6 (Magnusson-Kligman-Test)

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:

Test was not valid at time of test conduct

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-[(6-chloro-1,3,5-triazine-2,4-diyl)diimino]bis[5-hydroxy-6-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonic] acid, sodium salt

EC Number:

303-152-9

EC Name:

4,4'-[(6-chloro-1,3,5-triazine-2,4-diyl)diimino]bis[5-hydroxy-6-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonic] acid, sodium salt

Cas Number:

94158-79-9

Molecular formula:

C39H32ClN9O26S8.xNa C39H26ClN9Na6O26S8 as hexasodium salt

IUPAC Name:

sodium 5-[(4-chloro-6-{[8-hydroxy-3,6-disulfonato-7-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalen-1-yl]amino}-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-(2-{4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)naphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Details on test material:

Reactive Red 230

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Pirbright-White

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG
- Weight at study initiation: 240 to 288 g; mean: 257 g
- Housing: 5/cage
- Diet: Altromin 3112 guinea pig maintenance diet ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12. October to 27 November 1992

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

5% / 4 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% / 0.5 mL

Day(s)/duration:

Day 8 for 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 for 24 h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Determination of primary non-irritating concentration: 6
Determination of the intradermal tolerance: 3
Number of animals in attending group: 5
Number of animals in test group: 10 Number of animals in negative control group: 5

Details on study design:

RANGE FINDING TESTS:
- Determination of primary non-irritating concentration
For the determination of the primary non-irritant concentration, preparations of 1, 5, and 25% test substance (25% being the highest concentration that could be formulated) in isotonic saline were tested in a dermal-occlusive test for primary skin irritation in the intact skin on the flanks of two guinea pigs. For this purpose, the hair on the flanks of the animals was removed mechanically and 0.5 mL of the test substance preparation was applied to a 2 x 2 cm² cellulose patch, which was then fixed to the 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. No signs of irritation occurred after application of the different test concentrations to intact skin; a concentration of 25% test substance in isotonic saline was therefore chosen for the challenge on Day 22, as it was considered adequate as highest non-irritating concentration for epidermal challenge purposes.

- Determination of the intradermal tolerance
The determination of the concentration for the intradermal induction was performed with concentrations of 0.2, 1, and 5% of the test substance in isotonic saline. Each of the concentrations was administered twice by intra-dermal injection to 3 guinea pigs. The injection sites (sites 1, 2 and 3) were all within a dorsal area measuring 2 x 4 cm² in the vicinity of the shoulder.

site application volume in mL concentration 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 injections of the 5% preparation caused very slight up to well defined oedema and encrustations at the application site. After application of the 1% and 0.2% preparations very slight oedema occurred one day after injection. In all cases evaluation of erythema was not possible due to red discoloured skin. Based on this pre-liminary test, a 5% preparation was selected for the intra-dermal injections in the main test.




MAIN STUDY
A. INTRADERMAL INDUCTION
- No of Injections: 2 x 3 preparations: 50% FCA, 5% TS in 0.9% NaCl, 5% TS in 50% FCA - treatment group
50% FCA, 0.9% NaCl, 50% FCA - control and attending group
- Exposure period: Injection on Day 1, observation Day 1 to Day 8
- Site: shoulder

B. DERMAL INDUCTION EXPOSURE
- No. of exposures: one
- Exposure period: 48 hours
- Test groups: TS in 0.9% NaCl
- Control group: 0.9% NaCl
- Site: shoulder
- Frequency of applications: single
- Duration: Day 8 to Day 22
- Concentrations: 25%

The epidermal induction takes place on a skin area which was damaged by intra-dermal injections with Freund’s adjuvant and/or test substance in Freund’s adjuvant. For epidermal induction treatment, 0.5 mL of the 25.0% test substance preparation was applied on a 2 x 4 cm² cellulose patch to cover the area where the intra-dermal injections have been placed, i.e. on pre-damaged skin treated with Freund’s adjuvant. Treatment of the administration site with Freund's Adjuvant can lower the threshold value for primary irritation, hence the concentration determined for epidermal induction on pre-damaged skin and epidermal challenge on intact skin was the same. The intradermal injections with Freund's Adjuvant (with and without test substance) caused moderate oedema. Well defined up to moderate erythema was observed at the application sites without test sub-stance, whereas evaluation of erythema formation was not possible at the sites treated with the test compound in Freund's Adjuvant due to red discolouration of the skin. Additionally, the application sites treated with the test substance in Freund's Adjuvant were indurated and encrusted. The application sites treated with the test substance in the vehicle exhibited very slight oedema as well as encrusted skin. Intradermal applications of the vehicle caused no signs of irritation. During the main test, after the removal of the patch for epidermal induction on Day 10, erythema and oedema, indurated and encrusted skin as well as necrosis were observed in the control group at the sites treated previously with Freund's Adjuvant. The injection sites treated with the test substance in the vehicle and with the vehicle alone showed no oedema; however, due to the red discoloured skin noted in the animals of the treatment group erythema evaluation was not possible. Due to the skin reactions observed, the concentrations of 5% for intradermal and 25% for epidermal induction were adequate concentrations to cause “mild-to-moderate skin irritation” for induction purposes.

C. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 22 (15 for attending group)
- Exposure period: 24 hours
- Test groups: TS in isotonic saline
- Control group: TS in isotonic saline
- Site: left flank
- Concentrations: 25%
- Evaluation (hr after challenge): 24 and 48 hours

Challenge controls:

In addition to the control group, 5 further guinea pigs (attending group) were used to confirm that challenge exposure with 25% TS would not lead to dermal irritation in animals pre-treated with 50 % FCA.

Positive control substance(s):

yes

Remarks:

bi-annual validation of assay

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

skin slightly reddish stained by TS

Remarks on result:

no indication of skin sensitisation

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:

skin slightly reddish stained by TS

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

skin slightly reddish stained by TS

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:

skin slightly reddish stained by TS

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

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 moderate oedema. Well defined up to moderate erythema was observed at the application sites without test substance, whereas evaluation of erythema formation was not possible at the sites treated with the test compound in Freund's Adjuvant due to red discolouration of the skin. Additionally, the application sites treated with the test substance in Freund's Adjuvant were indurated and encrusted. The application sites treated with the test substance in the vehicle exhibited very slight oedema as well as encrusted skin. Intradermal applications of the vehicle caused no signs of irritation. The application sites treated with the test substance were discoloured red.

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

After the removal of the patch at day 10, erythema and oedema, indurated and encrusted skin as well as necrosis were observed at the sites previously treated with Freund's Adjuvant. The injection sites treated with the test substance in the vehicle and with the vehicle alone showed no signs of irritation. Additionally, red 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 in the control and treatment group 24 and 48 hours after removal of the occlusive bandage. The treated area was discoloured slightly red.

 

Assessment

The epidermal induction takes place on a skin area which was damaged by intra-dermal injections with Freund’s adjuvant and/or test substance in Freund’s adjuvant. For epidermal induction treatment, 0.5 mL of the 25.0% test substance preparation was applied on a 2 x 4 cm² cellulose patch to cover the area where the intradermal injections have been placed, i.e. on pre-damaged skin treated with Freund’s adjuvant. Treatment of the administration site with Freund's Adjuvant can lower the threshold value for primary irritation, hence the concentration determined for epidermal induction on pre-damaged skin and epidermal challenge on intact skin was the same. The intradermal injections with Freund's Adjuvant (with and without test substance) caused moderate oedema. Well defined up to moderate erythema was observed at the application sites without test substance, whereas evaluation of erythema formation was not possible at the sites treated with the test compound in Freund's Adjuvant due to red discolouration of the skin. Additionally, the application sites treated with the test substance in Freund's Adjuvant were indurated and encrusted. The application sites treated with the test substance in the vehicle exhibited very slight oedema as well as encrusted skin. Intradermal applications of the vehicle caused no signs of irritation. During the main test, after the removal of the patch for epidermal induction on Day 10, erythema and oedema, indurated and encrusted skin as well as necrosis were observed in the control group at the sites treated previously with Freund's Adjuvant. The injection sites treated with the test substance in the vehicle and with the vehicle alone showed no oedema; however, due to the red discoloured skin noted in the animals of the treatment group erythema evaluation was not possible. Due to the skin reactions observed, the concentrations of 5% for intradermal and 25% for epidermal induction were adequate concentrations to cause “mild-to-moderate skin irritation” for induction purposes.

For the determination of the primary non-irritant concentration, preparations of 1, 5, and 25% test substance (25% being the highest concentration that could be formulated) in isotonic saline were tested in a dermal-occlusive test for primary skin irritation in the intact skin on the flanks of two guinea pigs. For this purpose, the hair on the flanks of the animals was removed mechanically and 0.5 mL of the test substance preparation was applied to a 2 x 2 cm² cellulose patch, which was then fixed to the 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. No signs of irritation occurred after application of the different test concentrations to intact skin; a concentration of 25% test substance in isotonic saline was therefore chosen for the challenge on Day 22. Hence, the concentration of 25% test substance was adequate as highest non-irritating concentration for epidermal challenge purposes.

 

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-Rot F-67 787 FW showed no evidence for sensitizing properties

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

There was no evidence of a positive reaction in animals after challenge treatment with Reaktiv-Rot F-67787 FW in the present study.
According to the classification criteria of Directive 83/467/EEC, Reaktiv-Rot F-67787 FW is not sensitizing in the guinea pig maximization test and therefore not subject to labelling requirements.

Executive summary:

The skin sensitizing potential of Reaktiv-Rot F-67787 FW was examined in female guinea pigs with the maximization test.

Intradermal induction was performed using 5% Reaktiv-Rot F-67787 FW in 0.9 % NaCl.solution.

Dermal induction and challenge treatment were carried out with 25 % Reaktiv-Rot F-67787 FW in 0.9 % NaCl.solution.

Based on the results of the present study, Reaktiv-Rot F-67787 FW showed no evidence for sensitizing properties and is not subject to labelling requirement.