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Skin sensitisation

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Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 January 1998 to 27 February 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to recent EU & OECD test guidance in compliance with GLP.
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
Pirbright-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: HARLAN WINKELMANN
- Weight at study initiation: mean - 366g (320 - 399g)
- Housing: in fully air-conditioned rooms in macrolon cages (type 4) on soft wood granulate in groups of 5 animals
- Diet (e.g. ad libitum): ssniff® Ms-H (V2233), ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 (± 3°C)
- Humidity (%): 50 (± 20 %)
- Photoperiod (hrs dark / hrs light): 12 hours daily

Route:
intradermal
Vehicle:
water
Remarks:
deionizied
Concentration / amount:
The following preparations were used for the intradermal injections:

Control group
1.) 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.
2.) Deionized water
3.) 50 % Freund's Complete Adjuvant emuision mixed with an equal volume of the vehicle

Treatment group
1.) 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.
2.) 5 % Reaktivgelb FD 08064 in deionized water
3.) 5 % Reaktivgelb FD 08064 in a 50 % Freund's Complete Adjuvant emulsion

For the intradermal injections of the test substance in 50 % Freund's adjuvant, Reaktivgelb FD 08064 was dissolved in deionized water and then mixed with an equal volume of Freund's Original Adjuvant [percentages w/v].

Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
deionizied
Concentration / amount:
The following preparations were used for the intradermal injections:

Control group
1.) 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.
2.) Deionized water
3.) 50 % Freund's Complete Adjuvant emuision mixed with an equal volume of the vehicle

Treatment group
1.) 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.
2.) 5 % Reaktivgelb FD 08064 in deionized water
3.) 5 % Reaktivgelb FD 08064 in a 50 % Freund's Complete Adjuvant emulsion

For the intradermal injections of the test substance in 50 % Freund's adjuvant, Reaktivgelb FD 08064 was dissolved in deionized water and then mixed with an equal volume of Freund's Original Adjuvant [percentages w/v].

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. In case of a questionable result additional animals will be tested to give a total of 20 test and 10 control animals.
Details on study design:
RANGE FINDING TESTS:

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 two guinea pigs:

25.0 % in deionized water
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.

Determination of the tolerance of the intradermal injections

To determine the tolerance of intradermal injections, each of the following prepara¬tions was administered twice by intradermal injection to 2 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.
24, 48, 72 and 96 hours after administration the injection sites were examined for local tolerance.


MAIN STUDY

A. INDUCTION EXPOSURE
- No. of exposures: 6
- Exposure period: Intradermal 7 days, dermal 48 hours
- Test groups: 50% Freund's Adjuvant, 5% substance in deionised water, 5% substance with 50% Freund's Adjuvant
- Control group: 50% Freund's Adjuvent, deinioised water, equal volume of deionised water with 50% Freund's Adjuvent
- Site: Within dorsal area
- Frequency of applications: once

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 22
- Exposure period: 24 hours
- Test groups: Test substance
- Control group: Test substance
- Site: Left flank
- Concentrations: 5.0%
- Evaluation (hr after challenge): 24 & 48 hours

Main test

Chronological description of the test procedure indicating the day, at which procedure was carried out, on the left margin of the page.
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.

Treatment group:

site appl. vol. vehicle
1 2x0.1 ml' - 50 % Freund's Adjuvant
2 2x0.1 ml 5% substance in deionized water
3 2x0.1 ml 5 % substance in 50 % Freund's Adjuvant

Control group:
site appl. Vol. vehicle
1 2 x 0.1 ml 50 % Freund's Adjuvant
2 2x0.1 ml deionized water
3 2 x 0.1 ml equal volume of deionized water and 50 % Freund's Adjuvant

The administration area was examined for local tolerance. Any systemic toxic effects were recorded.

Dermal induction treatment

An amount of 0.5 ml of the test substance preparation (treatment group) or the vehicle (control group) was administered to a 2 x 4 cm cellulose patch. This patch covered the area where the intradermal injection had been placed. The administration area was then kept for 48 hours under an occlusive bandage with an impermeable film and an elastic bandage.
Treatment group:25.0 % test substance in deionized water
Control group: deionized water
Dermal challenge treatment
One area of approx. 5 x 5 cm on the left flank was shaved mechanically.

An amount of 0.5 ml of the test substance preparation was administered to a 2 x 2 cm cellulose patch. The administration area was then kept for 24 hours under an occlusive bandage with an impermeable film and an elastic bandage.

Treatment and control group (left flank): 5.0 % Reaktivgelb FD 08064 in deionized water
After treatment, the occlusive bandage removed. Any remnants of the test substance were carefully washed off with warm water. Examination of the skin was undertaken approx. 24 and 48 hours after removal of the patches. Body weights of the test animals determined.
Challenge controls:
As above.
Positive control substance(s):
yes
Remarks:
Freund's Complete Adjuvant
Vehicle:
other: Not applicable
Positive control results:
Intradermal induction treatment

The intradermal injections with Freund's Adjuvant (with and without test substance) caused severe erythema and oedema as well as indurations and encrustations. Additionally, the injections sites treated with the test substance in Freund's Adjuvant showed open wounds and necrosis.

Dermal induction treatment

Control group: After the removal of the patches at day 10, severe erythema and oedema, indurated and encrusted skin as well as necrosis were observed at the sites previously treated with Freund's Adjuvant.
Treatment group: After the removal of the patches at day 10, severe erythema and oedema, indurated and encrusted skin as well as necrosis and open wounds were observed at the sites previously treated with Freund's Adjuvant.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Skin reddening
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Skin reddening.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Skin reddening
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Skin reddening.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Skin reddening
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Skin reddening.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Skin reddening
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Skin reddening.
Interpretation of results:
not sensitising
Remarks:
Migrated information
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.

Based on the results of this study Reaktivgelb FD 08064 showed no evidence for sensitizing properties.
Executive summary:

Study conducted to EU test guidance 96/54/EEC part B6 and OECD test guideline 406 in compliance with GLP.

The substance showed no evidence for sensitizing properties.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Sensitisation was assessed using the Magnusson and Kligmann sensitisation assay. During the induction phase of the study, strong irritation reactions were noted in both control and test groups. At the challenge stage, no signs of irritation were observed in the control and the treatment group 24 and 48 hours after removal of the occlusive bandage.

The substance elicited no allergenic response. Sensitisation rate: 0%

The substance is not considered to be a skin sensitiser.


Migrated from Short description of key information:
The substance is not considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Additional information:

The registered chemical is a reactive dye. For this class of dyes it was generally agreed between the members of the Ecological and Toxicological Association of Dyes and Organic Pigments Manufacturers (ETAD)that a possible risk for respiratory sensitisation for workers exists at high exposure.However the following should be noted:

 

  1. For the substance no history of respiratory problems, such as occupational asthma, is associated with the manufacture and use of the specific substance.
  2. Due to the granular form of the substance (spay dried in closed system from aqueous solution directly after synthesis) no risk for inhalative exposure arises.

The potential to cause respiratory sensitisation is therefore not considered to be applicable for this substance.

No evidence of respiratory sensitisation was noted in any of the studies conducted, and it is proposed that the substance is not a respiratory sensitiser.

Justification for classification or non-classification

The above study has been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP an in compliance with agreed protocols. sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification for sensitisation effects is therefore required.