REAKTIV-ORANGE DYPR 1410

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 November 1998 to 25 December 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

EEC-Guideline B.6 "Acute Toxicity Sensitization of the Skin" of the Directive 96/54/EEC: Commission Directive of July 30, 1996 adapting to technical progress for the twenty-second time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labeling of dangerous substances

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted July 17, 1992

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:

-

EC Number:

432-080-1

EC Name:

-

Molecular formula:

Hill formula: C18H15N3Na2O9S3 CAS formula: C18H17N3Na2O9S3.2Na

IUPAC Name:

Disodium 4-amino-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-1-sulfonate

Test material form:

solid: particulate/powder

Remarks:

microgranulate or well dedusted powder

Details on test material:

Name: Reaktiv-Orange DYPR 1410

In vivo test system

Test animals

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

female

Details on test animals and environmental conditions:

Species / sex: Pirbright-White guinea pig / female
Strain: HsdPocDH
Origin: HARLAN WINKELMANN Gartenstr. 27 D-33178 Borchen SPF breeding colony
Body weight at start of study: mean = 391g (=100 %)
min = 329g (-15.6%)
max = 417g (+6.7%)
n = 15
Randomization: Randomization scheme 98.0744
Animal maintenance: in fully air-conditioned rooms in macrolon cages (type 4) on soft wood granulate in groups of 5 animals

Room temperature: 20 ± 3°C
Relative humidity: 50 ± 20 %
Lighting time: 12 hours daily
Acclimatization: at least 7 days

Food: ssniff® Ms-H (V2233), ad libitum
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:

water

Concentration / amount:

Appl.vol. Conc. Vehicle
2 x 0.1ml - 50% Freund’s Adjuvant
2 x 0.1ml 5% Substance in deionised water
2 x 0.1ml 5% Substance in 50% Freund’s Adjuvant

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:

water

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

Challenge 1

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

25% /0.5 mL

Day(s)/duration:

Day 22 for 24 h

Adequacy of challenge:

highest non-irritant concentration

Challenge 2

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

25% / 0.5 mL

Day(s)/duration:

Day 29 for 24 h

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Test group Number of animals
Determination of the primary non-irritant concentration 3
Determination of the tolerance of the intradermal injections 2
Control group 5
Treatment group 10

10 animals in the treatment group and 5 animals in the control group were used. One animal of the treatment group were found dead at day 10 of the study.

Details on study design:

The following preparations were used for the intradermal injections:
Control group:
50 % Freund's Complete Adjuvant emulsion
Original Freund's Complete Adjuvant (Sigma Chemical Company) was mixed immediately before use with an equal volume of deionized water.
Deionized water
50 % Freund's Complete Adjuvant emulsion mixed with an equal volume of the vehicle

Treatment group:
50 % Freund's Complete Adjuvant emulsion
Original Freund's Complete Adjuvant (Sigma Chemical Company) was mixed immediately before use with an equal volume of deionized water.
5 % Reaktiv-Orange DYPR 1410 in deionized water
5 % Reaktiv-Orange DYPR 1410 in a 50 % Freund's Complete Adjuvant emulsion

For the intradermal injections of the test substance in 50 % Freund's adjuvant, Reaktiv-Orange DYPR 1410 was dissolved in deionized water and then mixed with an equal volume of Freund's Original Adjuvant [percentages w/v].
For the dermal treatments, Reaktiv-Orange DYPR 1410 was dissolved in deionized water [percentages w/v].
__________________________________________________________________________________

RANGE FINDING TESTS:

1. Determination of the primary non-irritant concentration
Prior to the determination of the primary non-irritation concentration in a dermal-occlusive test the animals received 4 intradermal injections of a 50% Freund's Complete Adjuvant emulsion (4 x 0.1 ml) into the dorsal area, since Freund's Complete Adjuvant may lower the threshold of primary irritation. Thereafter, each of the following test concentrations was administered to the flanks of three guinea pigs:
- 25.0 % in deionized water - max concentration that can be formulated
- 5.0 % in deionized water
- 1.0 % in deionized water

The hair on the flanks of the animals was removed mechanically. 0.5 ml of the test substance preparation was administered to a 2 x 2 cm cellulose patch, which was 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 administration of the different test concentrations.
Based on these results, a concentration of 25 % Reaktiv-Orange DYPR 1410 in deionized water was chosen for the challenge on Day 22 as the highest concentration that could be formulated and did not cause irritation in intact skin.

2. Determination of the tolerance of the intradermal injections
To determine the tolerance of intradermal injections, each of the following preparations was administered twice by intradermal injection to 2 guinea pigs.
site appl. vol. conc. vehicle
1 2 x 0.1 ml 5.0 % deionized water - max concentration that can be applied by injection
2 2 x 0.1 ml 1.0 % deionized water
3 2 x 0.1 ml 0.2 % deionized water

The injection sites (sites 1, 2 and 3) were all within a dorsal area measuring 2 x 4 cm in the vicinity of the shoulders. 24, 48, 72 and 96 hours after administration the injection sites were examined for local tolerance.
The intradermal injections with the 5 % and 1% preparation caused clear oedema and intradermal injections with the 0.2 % preparation caused slight oedema. Erythema was not perceptible, because intradermal injections of all test preparations caused red discoloration of the skin.
Based on this preliminary test, a 5 % preparation was selected for the intradermal injections in the main test as the maximum concentration that could be applied and caused clear irritation without causing systemic effects.

According to OECD TG 406, the test animals are initially exposed to the test substance by intradermal injection and/or epidermal application (induction exposure). The concentration of test substance used for each induction exposure should be well-tolerated systemically and should be the highest to cause mild-to-moderate skin irritation. The concentration used for the challenge exposure should be the highest non-irritant dose. The appropriate concentrations can be determined from a pilot study using two or three animals. Consideration should be given to the use of FCA-treated animals for this purpose.
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. As treatment of the administration site with Freund's Adjuvant can lower the threshold value for primary irritation, the concentrations determined for epidermal induction on pre-damaged skin and epidermal challenge on intact skin were the same.
The intradermal injections with Freund's Adjuvant, without test substance, caused severe erythema and oedema as well as indurations and encrustations. The administration sites treated with the test substance in deionized water and Freund's Adjuvant showed slight oedema and clear oedema, respectively. Intradermal injections of the vehicle alone exhibited no signs of irritation.
After the removal of the patches on Day 10, clear erythema and oedema, indurated and encrusted skin as well as necrosis were observed at the sites previously treated with Freund's Adjuvant, without test substance. The administration sites pre-treated with the test substance in deionized water and Freund's Adjuvant showed slight oedema and clear oedema, respectively. Erythema was not assessable, because the administration sites treated with test preparations were discoloured red.
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.

__________________________________________________________________________


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: one intradermal, one epidermal
- Exposure period: Day 1, Day 8 for 48 h
- Test groups: 1
- Control group: 1
- Site: dorsal neck
- Frequency of applications: single
- Duration: 48 h epidermal
- Concentrations: 5% intradermal, 25% epidermal

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: Day 22 and Day 29
- Exposure period: 24 h each
- Test groups: 1
- Control group: 1
- Site: left flank
- Concentrations: 25%
- Evaluation (hr after challenge): 24 and 48

First challenge treatment
24 and 48 hours after removal of the occlusive bandage no skin reactions were observed in the treatment and control group. Also challenge treatment did not cause any irritations.

Second challenge treatment
24 and 48 hours after removal of the occlusive bandage no skin reactions were observed in the treatment and control group. Also challenge treatment did not cause any irritations.

Positive control substance(s):

yes

Remarks:

Benzocain; periodically conducted positive control test

Results and discussion

Positive control results:

Testing for sensitizing properties of Benzocain was performed in female guinea pigs according to the method of MAGNUSSON & KLIGMAN. This study is conducted periodically, and used as supporting evidence to the fact that the study methodology and approach is appropriate.
Intradermal induction was performed using 1 % Benzocain in sesame oil DAB 10. Dermal induction and challenge treatment were carried out with 25 % Benzocain in sesame oil DAB 10.

After the second challenge treatment four animals of the treatment group (40 %) showed a positive reaction during the observation period. The results obtained are typical for weak sensitizers.

In vivo (non-LLNA)

Results

Results 1

Reading:

rechallenge

Hours after challenge:

24

Group:

positive control

Dose level:

25%

No. with + reactions:

4

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Results 2

Reading:

rechallenge

Hours after challenge:

48

Group:

positive control

Dose level:

25%

No. with + reactions:

4

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

25%

No. with + reactions:

1

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

25%

No. with + reactions:

1

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Results 6

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

9

Clinical observations:

one animal died on Day 10 without signs of toxicity

Remarks on result:

no indication of skin sensitisation

Results 7

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

9

Remarks on result:

no indication of skin sensitisation

Results 9

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Results 10

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

9

Remarks on result:

no indication of skin sensitisation

Results 11

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

25%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Results 12

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

9

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

First challenge treatment – control and treatment group

25% Reaktiv-Orange DYPR 1410 in deionised water

Treated area; left flank

 

Scoring of dermal reactions

Time of observation: approx. 24 hours after removal of the patches
Control animal No:	1		2		3		4		5
Value	0		0		0		0		0
Treated animal No.:	6		7		8		9		10		11		12*	13	14	15
Value	0		0		0		0		0		0		-	0	0	0
Time of observation: approx. 48 hours after removal of the patches
Control animal No.:		1		2		3		4		5
Value		0		0		0		0		0
Treated animal No.:		6		7		8		9		10		11	12*	13	14	15
Value		0		0		0		0		0		0	-	0	0	0

*Animal was found dead on day 10 of the study.

 

Second challenge treatment – control and treatment group

25% Reaktiv-Orange DYPR 1410 in deionised water

Treated area; left flank

 

Scoring of dermal reactions

Time of observation: approx. 24 hours after removal of the patches
Control animal No:	1		2		3		4		5
Value	0		0		0		0		0
Treated animal No.:	6		7		8		9		10		11		12*	13	14	15
Value	0		0		0		0		0		0		-	0	0	0
Time of observation: approx. 48 hours after removal of the patches
Control animal No.:		1		2		3		4		5
Value		0		0		0		0		0
Treated animal No.:		6		7		8		9		10		11	12*	13	14	15
Value		0		0		0		0		0		0	-	0	0	0

*Animal was found dead on day 10 of the study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, none of nine 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-Orange DYPR 1410 showed no evidence for sensitizing properties

Executive summary:

The present study was conducted in compliance with EEC-Guideline B.6 "Acute Toxicity Sensitization of the Skin" of the Directive 96/54/EEC and OECD-Guideline for testing of chemicals, 406 "Skin Sensitization". This study was conducted in compliance with GLP.

Testing for sensitizing properties of Reaktiv-Orange DYPR 1410 was performed in female guinea pigs according to the method of MAGNUSSON & KLIGMAN.

Intradermal induction was performed using 5 % Reaktiv-Orange DYPR 1410 in deionized water. Dermal induction and challenge treatments were carried out with 25 % Reaktiv-Orange DYPR 1410 in deionized water. 

The validity of the test system is confirmed by the periodically conducted positive control test using benzocain for the maximization test (report number 98.0490, dated June 30, 1998; Hoechst Marion Roussel, Preclinical Development Germany, Drug Safety). 

One animal of the treatment group was found dead at day 10 of the study. But no substance related toxic effects was noted. 

Under the conditions of the present study, none of nine 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-Orange DYPR 1410 showed no evidence for sensitizing properties.