Reactive Orange 16

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

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

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

Route:

intradermal

Vehicle:

physiological saline

Remarks:

and freund's complete adjuvant

Concentration / amount:

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

Route:

epicutaneous, occlusive

Vehicle:

physiological saline

Remarks:

and freund's complete adjuvant

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

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.

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.

Reading:

1st reading

Group:

negative control

Remarks on result:

not measured/tested

Reading:

1st reading

Group:

positive control

Remarks on result:

not measured/tested

Reading:

2nd reading

Group:

positive control

Remarks on result:

not measured/tested

Reading:

2nd reading

Group:

negative control

Remarks on result:

not measured/tested

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Reactive orange 16 is not an allergen on guinea pigs, 0% of positive skin reactions of a group of 20 animals.

Respiratory sensitisation

Link to relevant study records

Reference

Endpoint:

respiratory sensitisation: in vivo

Type of information:

other: read across from supporting substance

Adequacy of study:

key study

Study period:

11. Aug to 21. Oct. 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Principles of method if other than guideline:

According to the European Discussion Group of Inhalation Toxicologists (EDIT) referring to Botham P.A., Rattraya N.J., Woodcocka D.R., Walsha S.T. and Hexta P.M. "The induction of respiratory allergy in guinea-pigs following intradermal injection of trimellitic anhydride: a comparison with the response to 2,4-dinitrochlorobenzene ", Toxicology Letters, Volume 47, Issue 1, April 1989, Pages 25-39

GLP compliance:

yes

Species:

guinea pig

Strain:

other: Pirbright-White (HOE DHPK (SPFLac))

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG
- Age at study initiation: 3 to 5 weeks
- Weight at study initiation (mean): males: 250 g; females: 243 g
- Housing: groups of 4 animals
- Diet: Altromin 3112 ad libitum
- Water: tap ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 30 to 70
- Air changes (per hr): -
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 11-Aug-1993 To: 21-Oct-1993

Route of induction exposure:

intradermal

Route of challenge exposure:

inhalation

Vehicle:

other: intradermal: NaCl

Concentration:

males: 150, 210 mg/m³
females: 140, 180 mg/m³

No. of animals per dose:

Determination of the tolerance of intradermal injections: 2 animals
Determination of the primary non-irritant concentration (inhalation): 4 animais
Material Control Group: 8 animals
Test Group: 8 animals

Details on study design:

Animals of the test groups are treated intradermally with the test substance: 0.1 mL 1%, 5%, 30%
Animals of the material control groups are treated with the vehicle only.
Determination of the primary non irritant aerosol concentration: 30 to 200 mg/m³
All animals are challenged by inhalation after three weeks using a primary non irritating concentration of the test substance.
Allergic reactions in the test groups are assessed by changes of lung function parameters compared to the material control groups.

Challenge controls:

yes

Positive control substance(s):

not specified

Negative control substance(s):

not specified

Results:

Determination of the tolerance of intradermal injections:
Intradermal injection of 30% Remazol-Schwarz B in physiological saline caused encrustations and beginning necrosis at the application sites. Slight induration was observed after injection of the 5% formulation. The injection sites treated with 1% Remazol - Schwarz B showed no signs of irritation. Based on these results a 5% solution was chosen for intradermal induction at day 1.

Determination of the primary non-irritant aerosol concentration:
A slight increase in respiratory rate occurred during exposure to approx. 200 mg/mg air. No marked changes in respiratory rate were observed during exposure to approx. 160 mg Remazol-Schwarz B/m³. Therefore this concentration was chosen for inhalation exposure at challenge day 22.

Body weight gain and clinical signs:
The intradermal injections caused encrustations and indurations of the injection sites up to day 8 at the study. Body weight gains were not impaired.

Challenge treatment
– Lung function parameters
Questionable up to slight changes in the respiratory pattern were observed in all animals after onset of exposure. No significant differences were present between the animals of the material control groups and the animals of the test groups.
Based on the results of the present study Remazol-Schwarz B did not cause a significant allergic response after intradermal induction and respiratory challenge.

- Clinical signs of intoxication
No clinical signs of intoxication were observed.

- Autopsy findings
Autopsy of the animals revealed red patches on the lungs in one animal of each group, respectively.

Positive control results:

-

Negative control results:

-

Interpretation of results:

not sensitising

Conclusions:

Based on the results of the present study the substance did not cause a significant allergic response after intradermal induction and respiratory challenge.

Executive summary:

The substance was tested for respiratory sensitization in Pirbright White guinea pigs. Induction was carried out by intradermal injection of 0.1 ml test item (5% solution in physiological saline) at day 1. Animals were challenged at day 22 with approx. 140 - 210 mg Remazol-Schwarz B / m3 air. Evaluation of the particle size distribution of Remazol-Schwarz B aerosol revealed a mass median aerodynamic diameter (MMAD) of 1.8 micrometers and a geometric standard deviation of 1.9. The respiratory pattern changed slightly in some animals during challenge on day 22, but there were no marked differences between the animals induced with Remazol-Schwarz B and control animals, which received isotonic saline only on the day of induction. Based on the results of the present study the substance did not cause a significant allergic response after intradermal induction and respiratory challenge.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Similar substance 2 was tested for respiratory sensitization in Pirbright White guinea pigs. Induction was carried out by intradermal injection of 0.1 ml test item (5% solution in physiological saline) at day 1. Animals were challenged at day 22 with approx. 140 - 210 mg test item / m^3 air. Evaluation of the particle size distribution of test item aerosol revealed a mass median aerodynamic diameter (MMAD) of 1.8 micrometers and a geometric standard deviation of 1.9. The respiratory pattern changed slightly in some animals during challenge on day 22, but there were no marked differences between the animals induced with test item and control animals, which received isotonic saline only on the day of induction. Based on the results of the present study the test item did not cause a significant allergic response after intradermal induction and respiratory challenge.

Justification for classification or non-classification

According to EU Guidelines, Reactive Orange 16 was not sensitizing for skin and respiratory. However, literature data report on symptoms of respiratory allergy observed in workers with a high occupational exposure to reactive dyes.

Consequently, it was agreed between members of the ETAD to classify Reactive Orange 16 as skin and respiratory sensitizer.