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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:
15 March 1994 to 08 April 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU and OECD guidelines in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Guideline 84/449
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH
- Age at study initiation: 5 to 7 Weeks
- Weight at study initiation: Mean 331g (298 to 360g)
- Housing: 2 -3 animals per cage
- Diet (e.g. ad libitum): Altromin 3020, ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 40 - 70
- Air changes (per hr): 12 - 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours, artificial illumination from 6am to 6 pm

Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
Intradermal induction: 5% (=20 mg test compound/animal)
Topical induction: 50% (=250 mg test compound/animal)
Challenge: 50% (=250 mg test compound/animal) and 25% (=125 mg test compound/animal)
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
Intradermal induction: 5% (=20 mg test compound/animal)
Topical induction: 50% (=250 mg test compound/animal)
Challenge: 50% (=250 mg test compound/animal) and 25% (=125 mg test compound/animal)
No. of animals per dose:
One test substance group consisting of 20 experimental animals (No. 21 to 40) and two control groups consisting of 10 animals each (No. 1 to 10 and 11 to 20)
Details on study design:
Intradermal Induction
One day prior to application the skin on the dorsum and the flanks was shorn. Starting behind the nucha,three intradermal injections were administered on each side of and parallel to the spinal column.
The application volume per injection site was 0.1 ml, The animals of the three groups were treated as follows:

a) Test substance group

1. Injection site pair (cranial)
Freund's complete adjuvant (Difco Lab.) 1:1 diluted with physiological saline solution (sterile).
2. Injection site pair (medial)
Reactive Blue FC 75311 5%, formulated with physiological saline solution (sterile).
3. Injection site pair (caudal)
Reactive Blue FC 75311 5%, formulated in physiological saline solution and Freund's complete adjuvant in equal proportions.
b) Control groups
The animals of the control groups were treated like the animals of the test substance group, the formulation for the injection site pairs 2 and 3, however, contained no test compound but a corresponding amount of sterile physiological saline solution.

Topical Induction
One week after intradermal induction topical induction took place. On the eve, the areas to be treated were shorn, and a 10% preparation of sodium lauryl sulphate in Vaseline was spread on these areas. Hypoallergenic patches {2x4 cm) were applied between or on the injection sites; they were covered with aluminum foil and kept in place on the skin with Fermoflex adhesive tape (Transatlantic GmbH, Schwarzenbach).
The hypoallergenic patches were treated as follows:
a) Test substance group: 0.5 ml Reactive Blue 75311 50%
b) Control group: 0.5 ml physiological saline solution (sterile)
At the end of the 48-hour exposure period the substance residues were removed with tap water.

Topical Challenge of Sensitization
The topical challenge took place 3 weeks after intradermal induction. Meanwhile, the test compound concentration for challenge had been determined in a range-finding test on 5 guinea pigs treated like control animals during inductions.

One day prior to the treatment the animals were shorn on the dorsum and the flanks. For challenge a hypoallergenic patch saturated with 50% and 25% test compound formulation was placed on the left flank of the animals of the test substance group and the ist control group. The patch was fixed for 24 hours on the skin with Fermoflex adhesive tape. The patches were applied in both concentrations cranially or caudally - alternately from animal to animal. On the right flank two control patches were fixed, saturated only with sterile physiological saline solution.
The application volume was 0.5 ml in each case.

At the end of the exposure period, substance residues were removed with tap water. 21 hours later the skin of the animals was depilated in the region of the treated areas. Pilca cream (depilation cream for humans, containing salts of the thioglycolic acid; manufactured by Schwarzkopf GmbH, Hamburg) was used for that purpose.

Challenge controls:
Control groups
The animals of the control groups were treated like the animals of the test substance group, the formulation for the injection site pairs 2 and 3, however, contained no test compound but a corresponding amount of sterile physiological saline solution.
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole. See below under free text
Vehicle:
other: Not applicable
Statistics:
To assess if and to what extent sensitization occurred, the number of animals of the test substance group and the control group with a skin irritant reaction and the severity of the reactions were established and a comparison was made. The criterion for sensitization was whether the occurrence of skin irritant reactions was more frequent and severe for the animals of the test substance group than for the control group.
Positive control results:
As above.
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
Test substance patch, skin reddening.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: Test substance patch, skin reddening..
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
25%
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
Test substance patch, skin reddening.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 25%. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: Test substance patch, skin reddening..
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Control patch, skin reddening
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Control patch, skin reddening.
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Control patch, skin reddening
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Control patch, skin reddening.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Test substance patch, skin reddening
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: Test substance patch, skin reddening.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Test substance patch, skin reddening
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Test substance patch, skin reddening.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Control patch, skin reddening
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Control patch, skin reddening.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Control patch, skin reddening
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Control patch, skin reddening.
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Test substance patch, skin reddening
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Test substance patch, skin reddening.
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
50%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
Test substance patch, skin reddening
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 50%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: Test substance patch, skin reddening.
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Control patch, skin reddening
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Control patch, skin reddening.
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Control patch, skin reddening
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: Control patch, skin reddening.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Test substance patch, skin reddening
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: Test substance patch, skin reddening.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
50%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Test substance patch, skin reddening
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 50%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: Test substance patch, skin reddening.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Control patch, skin reddening
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Control patch, skin reddening.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Control patch, skin reddening
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Control patch, skin reddening.
Parameter:
SI
Remarks on result:
other: Not applicable.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Not applicable.

The incidences of skin reddenings after challenge are summarized in the following table-.

 

 

Test substance Group (20 animals)

1stControl group (10 animals)

Test substance patch

Control patch

Test substance patch

Control patch

Hours

48

72

Total

48

72

48

72

Total

48

72

Challenge 50%

0

1

1

0

0

2

1

2

0

0

Challenge 25%

1

2

2

0

0

1

0

1

0

0
Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The substance was found to be non-sensitising in the maximisation test.
Executive summary:

Reactive Blue FC 75311 was evaluated for potential skin sensitizing properties in a maximisation test as described by MAGNUSSON and KLIGMANN. The test was performed on female guinea pigs.

The test compound was formulated in physiological saline solution (sterile) as a suspension.

After challenge one animal (5%) and two animals (10%) of the test substance group reacted with skin reddening to the 50% and 25% test compound formulation, respectively. In the control group two animals (20%) and one animal (10%), respectively, showed skin reactions.

Thus, under the conditions of the maximization test, the test compound has no skin sensitizing potential.

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, irritation reactions were noted in both control and test groups. After the challenge exposure one animal (5%) and two animals (10%) of the test substance group reacted with skin reddening to the 50% and 25% test compound formulation, respectively. In the control group two animals (20%) and one animal (10%), respectively, exhibited skin reactions.

 

On the basis of the results, the substance cannot be considered to be a skin sensitiser.


Migrated from Short description of key information:
The test substance showed no evidence for sensitizing properties in guinea pigs.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

The registered chemical is a reactive dye. For this class of dyes it was generally agreed between the members of theEcological 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.


Migrated from Short description of key information:
Not assessed. 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 Klimish 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.