Reactive Orange 16

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 9,1986 to May 10,1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EC Directive 83/467/EEC

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

available test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of disodium 6-acetamido-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2-sulfonate and sodium 6-acetamido-4-hydroxy-3-{[4(vinylsulfonyl)phenyl]diazenyl}naphthalene-2-sulfonate

Molecular formula:

n.a.

IUPAC Name:

Reaction mass of disodium 6-acetamido-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2-sulfonate and sodium 6-acetamido-4-hydroxy-3-{[4(vinylsulfonyl)phenyl]diazenyl}naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

Pirbright-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF-Zucht
- Strain: Hoe DHPK (SPFLac)
- Weight at study initiation: mean: 256 g (= 100%); mean min: 238 g (-8%); mean max: 270 g (+4%); n=15
- Housing: in fully air-conditioned rooms in Makrolon cages (type 4) on softwood granules, in groups of 5 animals
- Diet: ERKA-mixed feed No. 8300 for guinea pigs and rabbits, ad libitum
- Water: tap water ad libitum in plastic bottles
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3°C
- Humidity: 50 ± 20 %
- Photoperiod: 12 hours cycle dark/light

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

physiological saline

Remarks:

and freund's complete adjuvant

Induction 2

Concentration / amount:

treatment group
25% Remazol Brilliant Orange 3R FW in 0.9% NaCl solution

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Remarks:

and freund's complete adjuvant

Challenge 2

Concentration / amount:

treatment group
25% Remazol Brilliant Orange 3R FW in 0.9% NaCl solution

No. of animals per dose:

Determination of the primary concentration: 15
Determining compatibility of intradermal dose: 3
Support Group: 5
Control group: 5
Treatment group: 10

Details on study design:

SUBSTANCE FORMULATIONS
The Freund's Adjuvant was mixed immediately before use with an equal volume of 0.9 % of NaCl solution. This 50 % Freund's Adjuvant preparation was injected intradermal to the animals.
For the dermal application Reaktiv-Orange F-64258 FW was diluted with 0.9% NaCl, for the intradermal injections with Pyrogen and isotonic solution of NaCl.
For the intradermal injections Freund's adjuvant was diluted with Remazol Brilliant Orange 3R FW 0.9% NaCl solution pyrogen (percentages in w/v) and this solution then diluted with an equal volume of original - mixed Freund's adjuvant .
In the maximization test concentrations can’t be standardized. The suitable concentrations would be determined in preliminary studies. The concentration used depones on the individual test phase.

DETERMINATION OF THE PRIMARY NON-IRRITANT CONCENTRATION
Each of the following test concentrations were administered (occlusive) to the left flank of two guinea pigs:
25 % in 0.9% of NaCl solution
10 % in 0.9% of NaCl solution
1 % in 0.9% of NaCl solution
The right and left flank of the animals was depilated mechanically. 0.5 ml of the test substance preparation was administered to a 2 x 2 cm cellulose patch, which was fixed to the left flank and covered occlusively for 24 hours with a bandage foil. 24 hours after removal of the patches, the treated skin areas were examined for erythema and edema.

RANGE FINDING TESTS:
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 shoulders.
site appl. vol. conc. vehicle
1 2x0.1 ml 5.0 % RemazolbrillantOrange 3R FW in 0.9% NaCl solution
2 2x0.1 ml 1.0 % RemazolbrillantOrange 3R FW in 0.9% NaCl solution
3 2x0.1 ml 0.2 % RemazolbrillantOrange 3R FW in 0.9% NaCl solution

MAIN STUDY
Guinea pigs were shaved meccanically over a dorsal area of 4 x 6 cm in the vicinity of the shoulders.

INTRADERMAL INDUCTION EXPOSUR from day 1
- Site: dorsal area of 2 x 4 cm
- Applications: Two intradermal injections per animal and 3 injection sites in a area of 2 x 4 cm. The injection sites were left uncovered.
Groups concentration
site appl. vol. conc. vehicle
1 2x0.1 ml 50 % Freund’s adjuvant solution
2 2x0.1 ml 5% RemazolbrillantOrange 3R FW in 0.9% NaCl solution
3 2x0.1 ml 5 % RemazolbrillantOrange 3R FW in Freund’s adjuvant solution
Control group
site appl. vol. conc. vehicle
1 2x0.1 ml 50 % Freund’s adjuvant solution
2 2x0.1 ml 0.9% NaCl solution
3 2x0.1 ml 50 % Freund’s adjuvant solution


DERMAL INDUCTION EXPOSUR from day 9
- Concentration: 0.5 ml of the test substance preparation
- Site: 2 x 4 cm
- Applications: By cellulose patch. This patch coovered the area were intradermal injection had been placed. The administration area was then Kept under an occlusive bandage cooverade with any impermeable film and an elastic bandage for 48 h
- Treatment group: 25.0 % Remazol-Brilliantorange 3R FW in 0.9% in NaCl solution
- Control and satellite group: 0.9% NaCl solution
Bandage was removed at day 11

CHALLENGE EXPOSURE from day 22
An area of approx. 5 x 5 cm on the left and right flank was shaved mechanically.
- Concentration: 0.5 ml of the test substance preparation
- Site: 2 x 2 cm
- Bandage: the administartion area was kept for 24 under an occlusive bandage covered with an impermeable film and an elastic bandage.

At day 23 the occlusive bandage was removed.

Challenge controls:

left flank: 1% Remazol Brilliant Orange 3R FW in 0.9% NaCl solution

Positive control substance(s):

no

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1% test item inside 0.9% NaCl solutiion

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1% test item inside 0.9% NaCl solutiion. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 2

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

1% test item inside 0.9% NaCl solution

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 1% test item inside 0.9% NaCl solution. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 3

Reading:

1st reading

Group:

negative control

Remarks on result:

not measured/tested

Results 4

Reading:

1st reading

Group:

positive control

Remarks on result:

not measured/tested

Results 5

Reading:

2nd reading

Group:

positive control

Remarks on result:

not measured/tested

Results 6

Reading:

2nd reading

Group:

negative control

Remarks on result:

not measured/tested

Any other information on results incl. tables

DETRMINATION OF THE PRIMARY NON-IRRITANT CONCENTRATION

After treatment with 25% substance, three of five animals showed very mild erythema.

In the treatment with the 10% preparation the substance produced very slight erythema in five animals.

Application of 1% concentration did not cause anyirritation.

The treatment with Freund's Complete Adjuvant may lower the threshold of primary irritation, determined in the preliminary test. Therefore, 5 animals of the satellite group, treated with Freund's Complete Adjuvant, received a dermal administration of a 1 % Remazol-BrillantOrange FW in 0.9% NaCl solution.

 

This animals showed no signs of irritation. Therefore, a concentration of 1% RemazolBrilliant Orange 3R FW in NaCl solution was chosen for dermal challenge treatment at day 22.

 

DETRMINATION OF THE TOLERANCE OF THE INTRADERMAL INJECTIONS

Th]e intradermal injections with the 5 % preparation caused slight swelling and the 1% formulation caused a perceivable swelling. Redness did not occur.The injectionof the 0.2% formulation resulted no irritant.

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

 

MAIN TEST

- 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 one case of marked redness and swelling of the injection site within one week.

Additionally, beginning on day 3 after administration the injection sites were indurated.

After the removal of the patches on day 11, the injection sites of the animals from the control and satellite group were slightly reddened, swollen, indurated and scabbed.

Due to a substance related discoloration of the skin evaluation of erythema formation was not possible in the treatment group; it could only swelling, hardening and sloughing observed.

The body weight gains of the animals were not impaired.

 

- Dermal challenge treatment: 24 hours after removal of the occlusive bandage both the animals in the control as well as the animals in the treatment group showed no irritation symptoms.

There was only a compound-related reddening of stubble, which made the assessment of barely perceptible erythema impossible.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The present study has shown that after the release treatment no animals has given positive response.
According to the evaluation criteria of Directive 83/467/EEC,Reactive Orange 16 in the maximization test is non-sensitizing.

Executive summary:

Reactive Orange 16 was tested for skin sensitization with White guinea pig on maximization test.

The intradermal induction treatment with 5% Reactive Orange 16 in the dermal induction treatment with 25% Reactive orange 16 in 0.9% NaCl solution and the release treatment with 1% Reactive Orange 16 in 0.9% NaCl solution. The study had showed that Reactive Orange 16 under criteria of the EC Directive 83/467/EEC must be assessed as non-sensitizing.