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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 31, 1999 to September 01, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Available data over 12 years old.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
EEC-Guideline B.6" 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
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
OECD-Guideline for testing of chemicals, 406 "Skin Sensitization", adopted July 17, 1992
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Available data +12 years old.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
No further details specified in the study report.

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: HsdPoc: DH
Origin: HARLAN WINKELMANN, Gartenstr. 27, D-33178 Borchen, SPF breeding colony

Body weight of the main test group at start of study:
mean = 372 g (= 100 %)
mm = 341 g (- 8.3 %)
max = 439g (+ 18.0%)
n = 15

Randomization: Randomization scheme 1999.0312
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)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: sesame oil
Concentration / amount:
5.0%
Day(s)/duration:
Administration Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
intradermal
Vehicle:
other: 50 % Freund's Complete Adjuvant emulsion
Concentration / amount:
5%
Day(s)/duration:
Administration Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: Sesame oil
Concentration / amount:
25.0%
Day(s)/duration:
Single treatment Day 8 / 48 h
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: Sesame oil
Concentration / amount:
25%
Day(s)/duration:
Day 22 / 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Control group: 5 animals
Treatment group: 10 animals
Determination of the primary non-irritant concentration: 3 animals
Determination of the tolerance of the intradermal injections: 2 animals
Details on study design:
Test procedure
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) sesame oil (Vehicle)
3) 50 % Freund's Complete Adjuvant emulsion mixed with an equal volume of the vehicle
Treatment group
1) 50 % Freunds 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 % Resolin Blau BBLS 200% in sesame oil
3) 5 % Resolin Blau BBLS 200% in a 50 % Freund's Complete Adjuvant emulsion
For the intradermal injections of the test substance in 50 % Freund's adjuvant, Resolin Blau BBLS 200% was suspended in Freund's Original Adjuvant and then mixed with an equal volume of deionized water [percentages w/v].

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 were administered to the flanks of two guinea pigs:
25.0 % in sesame oil
5.0 % in sesame oil
1.0 % in sesame oil
The hair on the flanks of the animals was removed mechanically. 0.5 ml 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 edema.

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. 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 test for the sensitizing properties
Chronological description of the test procedure indicating the day, at which procedure was carried out, on the left margin of the page.
Study day 0
The body weight of the animals was determined.
The guinea pigs were shaved mechanically over a dorsal area of 4 x 6 cm in the vicinity of the shoulders.

Study day 1: Intradermal induction treatment
Two intradermal injections per animal of the following preparations were applied. The injection sites (site 1, 2 and 3) were all within a dorsal area of 2 x 4 cm. The injection sites were left uncovered.
Treatment group:
site appl. vol. conc. preparation
1 2 x 0.1 ml - 50 % Freund's Adjuvant
2 2 x 0.1 ml 5.0% substance in sesame oil
3 2 x 0.1 ml 5.0% substance in 50 % Freund's Adjuvant
Control group:
site appl. Vol. preparation
1 2 x 0.1 ml 50 % Freund's Adjuvant
2 2 x 0.1 ml sesame oil
3 2 x 0.1 ml equal volume of sesame oil and 50 % Freund's Adjuvant

Study day 2-7
The administration area was examined for local tolerance. Systemic toxic effects were recorded, when apparent.

Study day 8: 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 under an occlusive bandage covered with an impermeable film and an elastic bandage for 48 hours.
Treatment group: 25.0 % test substance in sesame oil
Control group: sesame oil

Study day 10
Occlusive bandage was removed, irritant effects were recorded, when apparent.

Study day 11 - 21
No treatment of control or treatment group.
Test animals were kept under observation.

Study day 22: 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 covered with an impermeable film and an elastic bandage.
Treatment and control group (left flank):
25 % Resolin Blau BBLS 200% in sesame oil

Study day 23
Occlusive bandage was removed. Any remnants of the test substance were carefully washed off with warm water.

Study day 24
Examination of the skin approx. 24 hours after removal of the patches.

Study day 25
Examination of the skin approx. 48 hours after removal of the patches.
Body weight of the test animals was determined.

Evaluation
Erythema and edema are major clinical indicators of an allergic reaction. The decisive criterion for evaluation of the sensitizing properties of a test substance is the number of sensitized test animals, not the intensity of the dermal reaction.
The substance is considered to be sensitizing if 30 % or more of the animals in the treatment group definitely showed a positive skin reaction and at the same time no irritant effects have emerged in the control group.
Positive control substance(s):
yes
Remarks:
alpha-hexyl cinnamic aldehyde

Results and discussion

Positive control results:
80% positive animals

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
The treated animals showed no clinical signs of intoxication throughout the study.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
The treated animals showed no clinical signs of intoxication throughout the study.
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
The treated animals showed no clinical signs of intoxication throughout the study.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
The treated animals showed no clinical signs of intoxication throughout the study.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25%
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25%
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Body weight gains

Animal No.

Body weight at the start of the study [g]

Body weight at the end of the study [g]

Increase [%]

Control group

1

2

3

4

5

370

439

345

384

349

554

683

584

586

569

+ 50

+ 56

+ 69

+ 53

+ 63

Treatment group

6

7

8

9

10

393

379

374

365

341

619

539

603

568

566

+ 58

+ 42

+ 61

+ 56

+ 66

11

12

13

14

15

372

407

378

347

342

537

577

540

487

556

+ 44

+ 42

+ 43

+ 40

+ 63

 

Tables of the individual data

 

Determination of the primary non-irritant concentration – individual values

Treated area: left and right flank

Time of observation: approx. 24 hours after removal of the patches

Animal No.

Concentration [%]

Results

1

Left flank

25

0

1

Right flank

5

0

2

Left flank

25

0

2

Right flank

1

0

3

Left flank

5

0

3

Right flank

1

0

 

Determination of the tolerance if the intradermal injections – individual values

Time after injection

Findings

5 %

1 %

0.2 %

Day 1

Slight erythema, blue discoloured, erythema not assessable

Slight erythema, blue discoloured; erythema not assessable

Blue discoloured; erythema not assessable

Day 2

Clear edema, blue discoloured; erythema not assessable

Slight edema, blue discoloured; erythema not assessable

No findings

Day 3

Clear edema, partly indurated, blue discoloured; erythema not assessable

Slight edema, partly indurated, blue discoloured; erythema not assessable

No findings

Day 4

Clear edema, partly indurated, blue discoloured; erythema not assessable

Slight edema, partly indurated, blue discoloured; erythema not assessable

No findings

 

Challenge treatment – control and treatment group

25 % Resolin Blau BBLS 200% in sesame oil (day 22)

Treated area: left flank

 

Scoring of dermal reactions

Time of observation: approx. 24 hours after removal of the patches (day 24)

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

0

 

Time of observation: approx. 48 hours after removal of the patches (day 25)

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

0

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
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, the test substance showed no evidence for sensitizing properties.
The substance is not classified according to CLP criteria.
Executive summary:

Testing for sensitizing properties of Disperse Blue 165 was performed in female guinea pigs according to the method of MAGNUSSON & KLIGMAN.

 

lntradermal induction was performed using 5 % test substance in sesame oil. Dermal induction and challenge treatment were carried out with 25 % in sesame oil.

 

The validity of the test system is confirmed by the periodically conducted positive control test using alpha-hexyl cinnamic aldehyde for the maximization test (report number 99.0220, dated May 14, 1999; Hoechst Marion Roussel Deutschland GmbH, ProTox).

 

Based on the results of this study Dsiperse Blue 165 showed no evidence for sensitizing properties.

The substance is not classified accordingly to CLP criteria.