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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:
From April 9,1986 to May 10,1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report date:
1986

Materials and methods

Test guidelineopen allclose all
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
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study already available

Test material

Constituent 1
Chemical structure
Reference substance name:
Disodium 6-acetamido-4-hydroxy-3-[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2-sulphonate
EC Number:
243-653-9
EC Name:
Disodium 6-acetamido-4-hydroxy-3-[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2-sulphonate
Cas Number:
20262-58-2
Molecular formula:
C20H19N3O11S3.2Na
IUPAC Name:
disodium 6-acetamido-4-hydroxy-3-[(4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]naphthalene-2-sulfonate
Test material form:
solid: particulate/powder
Details on test material:
see below

In vivo test system

Test animals

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

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Remarks:
and freund's complete adjuvant
Concentration / amount:
5% / 4 * 0.1 mL
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
25% / 0.5 mL
Day(s)/duration:
Day 9 / 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:
physiological saline
Concentration / amount:
1% / 0.5 mL
Day(s)/duration:
Day 22 /24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Determination of the dermal induction and challenge concentration: 15
Determination of the intradermal concentration: 3
Challenge Control 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 the test substance was diluted with 0.9% NaCl, for the intradermal injections with pyrogen-free and isotonic solution of NaCl.
For the intradermal injections, the test substance was dissolved in Freund's adjuvant and diluted with pyrogen-free 0.9% NaCl solution (percentages in w/v) and this solution then diluted with an equal volume of original - mixed Freund's adjuvant .
In the maximization test concentrations cannot be standardized. The suitable concentrations would be determined in preliminary studies. The concentration used depends 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 5 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 of the test substance 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 % 0.9% NaCl solution
2 2x0.1 mL 1.0 % 0.9% NaCl solution
3 2x0.1 mL 0.2 % 0.9% NaCl solution

MAIN STUDY
Guinea pigs were shaved mechanically over a dorsal area of 4 x 6 cm in the vicinity of the shoulders.

INTRADERMAL INDUCTION EXPOSURE - 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% test substance in 0.9% NaCl solution
3 2x0.1 mL 5 % test substance 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 EXPOSURE - day 9
- Concentration: 0.5 mL of the 25% test substance preparation
- Site: 2 x 4 cm
- Applications: By cellulose patch. This patch covered the area were intradermal injection had been placed. The administration area was then kept under an occlusive bandage covered with any impermeable film and an elastic bandage for 48 h
- Treatment group: 25.0 % test substance in 0.9% in NaCl solution
- Control and satellite group: 0.9% NaCl solution
Bandage was removed at day 11

Day 15: Challenge of the Challlenge Control (satellite) group (same desing as day 22)

CHALLENGE EXPOSURE - 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 1% test substance preparation
- Site: 2 x 2 cm
- left flank: 1% test substance in 0.9% NaCl solution for control and treatment group
- Bandage: the administration 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.
Days 24 and 25: reading of the skin
Challenge controls:
left flank: 1% test substance in 0.9% NaCl solution
Positive control substance(s):
yes
Remarks:
biannual testing

Results and discussion

Positive control results:
valid

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
25%
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

DETRMINATION OF THE PRIMARY NON-IRRITANT CONCENTRATION

Treatment with a 25% substance preparation: very mild erythema in three of five animals

Treatment with a 10% substance preparation: very mild erythema in five animals

Treatment with a 1% substance preparation: no signs of irritation

DETRMINATION OF THE TOLERANCE OF THE INTRADERMAL INJECTIONS

The intradermal injections with the 5% preparation caused slight swelling and the 1% formulation caused a perceivable swelling. Redness did not occur. The injection of the 0.2% formulation resulted no irritation.

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.

Challenge control - treatment with a 1% substance preparation:no signs of irritation.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The present study has shown that after the challenge no animal has given positive response. Hence, the test substane is non-sensitizing in the maximization test.
Executive summary:

The test substance was tested for skin sensitisation in Pirbright White guinea pigs in the guinea pig maximisation test.

Intradermal induction treatment was performed with 5% test substance in 0.9% NaCl solution, dermal induction treatment with 25% test substance in 0.9% NaCl solution and dermal challenge treatment with 1% test substance in 0.9% NaCl solution.

The test has shown that the test substance is not sensitising in guinea pigs.