Black HM 2482

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 15th to May 23rd, 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted May 12, 1981

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

EWG Directive 84/449/ EWG, Amtsblatt der Europaeischen Gemein-schaften L 251, Jahrgang 27, 19. 9. 84. B.6. Akute Toxizitaet - Sensibilisierung der Haut / 113

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Guinea pig maximisation test was available

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

400-920-6

EC Name:

-

Cas Number:

89857-06-7

Molecular formula:

C50 H53 N11 O14 S

IUPAC Name:

5'-[2-(7-{2-[4-(2-{1'-[3-(dimethylazaniumyl)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-8-hydroxy-6-sulfonatonaphthalen-2-yl)diazen-1-yl]-6'-hydroxy-3,4'-dimethyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium bis(2-hydroxypropanoate)

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Remarks:

DUHA KFM

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Kieintierfarm Madoerin AG CH 4414 Fuellinsdorf / Switzerland
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 372 - 452 g males; 346 - 464 g females
- Housing: individually in Makrolon type-3 cages with standard softwood bedding
- Diet: peIIeted standard Kliba 342, Batch 21/85 and 22/85 guinea pig breeding/maintenance diet ad libitum
- Water: communlty tap water from Itingen, ad libitum
- Acclimation period: six days under test conditions after veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 10 %
- Air changes: 10-15 per hour
- Photoperiod: 12 hours artificial fluoscent light/12 hours dark, music/light period

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

other: FCA with physiological saline

Concentration / amount:

FCA, 50:50 with physiological saline

Induction 2

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

3%

Induction 3

Route:

intradermal

Vehicle:

other: FCA

Concentration / amount:

50:50 mixture of FCA

Induction 4

Route:

epicutaneous, semiocclusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

25%

Day(s)/duration:

Day 8

Challenge

Route:

epicutaneous, semiocclusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

25%

No. of animals per dose:

Test article group: 10 males/10 females
Negative control group: 5 males/5 females

Details on study design:

PRELIMINARY STUDY
The objective of this investigation was to identify irritant test article concentrations suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test article, by the topical route of administration, was identified for the challenge application.
The procedure employed for these investigation was as follows:
Intradermal injections:
Intradermal injections (0,1 mI/site) were made into the clipped flank of four guinea-pigs at concentrations of 5, 3, 1 and 0.5 %. of the test article in physiological saline. The resulting dermal reactions were assessed 24 hours later.
The following parameters were recorded:
Erythema (E) - 0 to 4 numerical scores
Edema (0) - 0 to 4 numerical scores
Diameter (D) - mm

Erythema and edema were assessed using the following arbitrary scales:
Erythema and eschar formation
No erythema 0
Slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate erythema 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4

Edema formation
No edema 0
Slight edema (barely perceptible) 1
Well-defined edema (edges of area well-defiend by definite raising) 2
Moderate edema (raised approximately 1 mm) 3
Severe edema (raised more than 1 mm and extending beyond the area of exposure) 4

Topical applications :
Patches of filter paper (2 x 2 cm) were saturated with concentrations of 10 and 25% of the test article in physiological saline and applied to the clipped and shaved flanks of each of four guinea-pigs tested concentrations. The patches were covered by a strip of aluminium foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. The dressings were removed after an exposure period of 24 hours and the reaction sites were assessed for erythema and edema, on a numerical basis according to the scale described above. Further examination of the sites were performed 24 and 48 hours after removal of the dressings.

MAIN STUDY
A. INDUCTION EXPOSURE

Intradermal injections:
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 4 cm area in the clipped region as follows:
1) Freunds' complete adjuvant 50:50 with physiological saline for injection.
2) The test article, diluted to 3% with physiological saline
3) The test article at the concentration used in (2), emulsified in a 50:50 mixture of Freunds' complete adjuvant, and the vehicle used in (2).

Topical applications:
0ne week after the injections, the scapular area was again clipped and shaved free of hair. A 4 x 4 cm patch of filter paper was saturated with the test article (25%) and placed over the injection sites of the test animals. The patch was covered by aIuminium foil and firmly secured by an elastlc plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were Ieft in place for approximately 48 hours.
The guinea-pigs of the control group received identical treatment without the test article.

B. CHALLENGE EXPOSURE
The test and control guinea-pigs were challenged two weeks after the topical induction application. Hair was clipped and shaved from a 5 x 5 cm area on the left flank of each guinea-pig. A 2 x 2 cm patch of filter paper was saturated with a non-irritant concentration (25%) of the test article and applied to the flank in a similar method to that used for the topical application.
The dressings wre removed approximately 24 hours later. The sites were assessed for erythema using the arbitrary scale. The control animals were treated with the vehicle alone.

Re-challenge
A second challenge was performed two weeks after the first challenge. The method was similar to that described for the first challenge with the exception that the right flanks of aII the guinea-pigs were used. The control animals were treated with the test article in the same concentration as the animals of the test group to avoid wrong positive results.


OBSERVATIONS
Mortality/Viability: once daily
Body weights: at pre-treatment start, start of application and end of test
Toxic Symptoms: daily

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

25

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

NEGATIVE CONTROL
No positive reactions were evident after the first and second challenge application.
TEST ARTICLE
After the first and second challenge application a bluish dicoloration was observed. No other symptoms or skin sensitizing effects were evident after the 24- and-l 48-hour reading.
VIABILITY/MORTALITY
Animal No. 74 (female) was replaced at day 1 of test due to incidental death. 
SYMPTOMS
LOCAL
Control group
Application area around the injections of series 1 and 3 were found to show wrythema and edema in any animal of the control group between days 2 and 4 of test. Starting with day 5 to 12 of test aditionally crusts were observed. Crusts were observed between days 13 and 22. Exfoliation was observed between days 23 to 26. These findings showed a clear healing tendency during the remaining test period.
Test article group
The animals of the test article-treated group showed the same reactions as described above.
SYSTEMIC
No systemic symptoms were observed.
BODY WEIGHTS
The body weight gain of all animals was not affected by the test procedure.

Applicant's summary and conclusion

Interpretation of results:

other: not classified for skin sensitisation according to the CLP Regulation (EC) No.1272/2008

Conclusions:

Non sensitizer.

Executive summary:

The allergenic potential of test substance was assessed in albino guinea pigs by the maximization-test of B. Magnusson and A.M. Kligman (1969), as per OECD guideline 406. Five males and five females were used as control group and 10 males and 10 females were used as test group. The highest non-irritating test article concentration used for challenge application was 25%. 
According to the procedures used in this experiment, no differences between the test group and the vehicle-treated controls were evident after epidermal challenge application of test item. The substance is considered to possess no skin sensitizing (contact allergenic) potential in albino guinea pigs. The incidence of positive reactions after epicutaneous challenge were as follows:
POSITIVE ERYTHEMA REACTIONS AFTER 1ST CHALLENGE PROCEDURE
                                                                   after 24 h                            after 48 h
                                                                        total                                    total
                                                              positive   negative                   positive   negative
Test substance                                            0            20                              0           20
(%) positive reaction/total animals            0                                              0
Vehicle control                                            0            10                               0           10
(%) positive reaction/total animals            0                                              0

After the second challenge application the following positive results were observed in the animals of the test article treated group:
after the 24-hour reading: 0 (0%)
after the 48-hour reading: 0 (0%)
No toxic symptoms were evident in the guinea pigs of either the control and test group.

In conclusion, 0% of the animals were positive after treatment with the non-irritating test substance concentration of 25%. Therefore the test article is considered to be a non sensitizer.