Disodium 5-(benzoylamino)-4-hydroxy-3-[[2-(2-methylphenoxy)phenyl]azo]naphthalene-2,7-disulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 7th to September 21st, 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The test results described do not totally comply with the specific testing guideline but they are sufficient to accept the data.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4100 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was already existing when LLNA was introduced in the Regulation

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 5-(benzoylamino)-4-hydroxy-3-[[2-(2-methylphenoxy)phenyl]azo]naphthalene-2,7-disulphonate

EC Number:

274-434-6

EC Name:

Disodium 5-(benzoylamino)-4-hydroxy-3-[[2-(2-methylphenoxy)phenyl]azo]naphthalene-2,7-disulphonate

Cas Number:

70210-37-6

Molecular formula:

C30H21N3O9S2.2Na

IUPAC Name:

disodium (E)-5-benzamido-4-hydroxy-3-((2-(otolyloxy)phenyl)diazenyl)naphthalene-2,7-disulfonate

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France (Saint-Aubin-Lès-Elbeuf, 76410 Cléon, France).
- Age at initiation of treatment: young adult (about 6 weeks old).
- Body weight range at initiation of treatment: 250 - 550 g.
- Diet: pelleted complete guinea-pig diet ad libitum
- Water: softened and filtered mains drinking water, ad libitum analysed at least once a year for chemical contaminants and at least twice a year for bacterial contaminants
- Caging: animals housed in groups according to EEC/86/609 in stainless steel mesh cages (internal dimensions 500 x 600 x 200 mm).

ENVIRONMENTAL CONDITIONS
- Housing: in an air-conditioned building (building G4)
- Temperature : 19 ± 3 °C
- relative humidity : ≥ 31 % R.H.
- air changes : ≥ 22 air changes per hour
- lighting cycle : 12 hours light (artificial)/12 hours dark

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

water

Induction 2

Concentration / amount:

PRELIMMINARY STUDIES:
- one injection of 0.1 ml per area and per animal, of the test article in a 5 % suspension or in a 1 % or 0.1 % (W/W) solution in water for injection.
- 0.5 ml per area and per animal of the test article in a 57 % or 28 % (W/W) paste in water for injection.
- 0.5 ml per area and per animal of the test article in a 10 % (W/W) suspension or in a 1 % (W/W) solution in water for injection.

MAIN STUDY:
- 0.5 ml per animal of the test article in a 1 % (W/W) solution in water for injection.

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

water

Challenge 2

Concentration / amount:

PRELIMMINARY STUDIES:
- one injection of 0.1 ml per area and per animal, of the test article in a 5 % suspension or in a 1 % or 0.1 % (W/W) solution in water for injection.
- 0.5 ml per area and per animal of the test article in a 57 % or 28 % (W/W) paste in water for injection.
- 0.5 ml per area and per animal of the test article in a 10 % (W/W) suspension or in a 1 % (W/W) solution in water for injection.

MAIN STUDY:
- 0.5 ml per animal of the test article in a 1 % (W/W) solution in water for injection.

No. of animals per dose:

• preliminary studies (minimum of 3) : 2 males, 2 non-pregnant females, per study.
• main study :
- control group -> 5 males, 5 nulliparous non-pregnant females.
- treated group -> 10 males, 10 nulliparous non-pregnant females. Two extra guinea-pigs (1 male and 1 female) will also be treated to allow for any possible non-treatment related deaths.
- option : positive control group -> 5 males, 5 nulliparous non-pregnant females.

Details on study design:

During induction, the applications were performed as follows :
• Treated group :
- By intradermal route: 3 series of 2 x 0.1 ml injections
Freund's complete adjuvant at 50 % (V/V) in an isotonic injectable solution;
test article in a 5 % (w/w) suspension in water for injection;
mixture 50/50 (V/V): test article in a 5 % (w/w) suspension in water for injection + Freund's complete adjuvant at 50 % (V/V) in an isotonic injectable solution, i.e. a final 2.5 % concentration of the test article .
During the preliminary study, injection of the test article in a 5 % suspension tinted the skin of the animals thus making observation of erythema impossible. No oedema was noted.
- By topical occlusive route for 48 hours, with 0.5 ml of the test article in a 57 % (w/w) paste in water for injection.
During the preliminary study, the test article tinted the skin of the animals thus making observation of erythema impossible. Nevertheless as no oedema was noted, a skin painting was performed during the main study on Day 8, with 0.5 ml of sodium lauryl sulphate at 10 % (w/w) in Codex paraffin to create irritation.
• Control group:
The intradermal injections and the topical occlusive application for 48 hours were carried out under the same conditions as in the treated group, water for injection replacing the test article.
The rest period was 11 days without treatment.
During the challenge, the topical occlusive application for 24 hours was performed in the treated group and in the control group with the test article in a 1 % (w/w) solution in water for injection and at the dose level of 0.5 ml. The cutaneous macroscopic examinations were performed 24 and 48 hours after removal of the patches to the challenge application site, according to the Magnusson & Kligman scale.
As the test article tinted the skin of the animals, thus making observation of erythema impossible, histopathological examinations of the skin were performed for all the animals of the treated and control groups (in half of them at 24 hours and in the other half at 48 hours).

Positive control substance(s):

yes

Remarks:

2,4-Dinitrochlorobenzene

Results and discussion

Positive control results:

80 to 100 % of sensitized animals are usually obtained.

In vivo (non-LLNA)

Results

Results 1

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1 % solution

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

Details are repotrted below

Remarks on result:

other: The full result table is attached below

Results 2

Group:

positive control

Remarks on result:

other: See table below

Results 3

Group:

negative control

Remarks on result:

other: See table below

Any other information on results incl. tables

Treated groups:

Biopsies performed at 24 hours						Biopsies performed at 48 hours
Animal (sex,number)	M62531	M62533	M62535	M62537	M62539	M62534 treated area	M62534 control area	M62536	M62538	M62540	M6262541
EPIDERMIS
Acanthosis	moderate	minimal	minimal	minimal	slight	slight	slight	minimal	slight	minimal	slight
Hyperkeratosis	moderate	slight	slight	moderate	moderate	moderate	moderate	marked	moderate	moderate	marked
Exocytosis	minimal (focal)	-	-	-	-	minimal (focal)	-	-	-	-	-
Spongiosis	minimal (basal)	-	-	-	minimal (basal)	minimal (focal)	-	-	-	-	-
DERMIS
Mononuclear cell infiltration	slight	slight	slight	slight	slight	slight	slight	slight (1)	slight	slight	slight
Folliculitis	-	-	-	-	minimal	-	-	-	-	-	-
Oedema	minimal	-	-	-	-	-	-	-	-	-	-

Biopsies performed at 24 hours							Biopsies performed at 48 hours
Animal (sex,number)	F62543	F62545	F62547 treated area	F62547 control area	F62549	F62551	F62542	F62544	F62546	F62548	F62550
EPIDERMIS
Acanthosis	slight	slight	minimal	slight	slight	moderate	slight	minimal	slight	minimal	slight
Hyperkeratosis	moderate	moderate	moderate	moderate	moderate	marked	moderate	moderate	moderate	moderate	moderate
Exocytosis	-	-	-	minimal (multifocal)	-	-	-	-	-	-	-
Spongiosis	-	-	-	minimal (basal)	-	-	-	-	-	-	-
Scab(s)	-	present	-	-	-	-	present	-	-	-	-
Ulceration	-	minimal (focal)	-	-	-	-	-	-	-	-	-
DERMIS
Mononuclear cell infiltration	slight	slight (1)	minimal	slight (1)	slight	slight	slight	slight	slight	slight	slight
Folliculitis	-	-	-	-	-	-	-	-	-	minimal	-
Oedema	-	-	-	-	-	-	-	-	-	-	-

Control group:

Biopsies performed at 24 hours						Biopsies performed at 48 hours
Animal (sex,number)	M62521	M62523	M62525	F62527	F62529	M62522	M62524	M62526	F62528	F62530
EPIDERMIS
Acanthosis	minimal	-	minimal	slight	slight	minimal	minimal	slight	minimal	slight
Hyperkeratosis	slight	slight	moderate	moderate	moderate	moderate	moderate	moderate	moderate	moderate
Exocytosis	-	-	-	-	minimal (focal)	minimal (focal) (1)	-	-	-	-
Spongiosis	-	-	-	-	minimal (basal)	slight (focal)	-	-	-	-
scab(s)	-	-	-	-	-	-	-	-	-	present
DERMIS
Mononuclear cell infiltration	minimal	minimal	minimal	slight	slight	slight	slight	slight	slight	slight
Folliculitis	-	-	-	-	-	-	-	minimal (focal)	-	minimal (focal)
Oedema	-	-	-	-	-	-	-	-	-	-

(1) with some polymorphs

Male no. 62534 showed on the treated area a minimal focus of spongiosis associated with exocytosis which could be due to cell mediated delayed hypersensitivity. Other changes seen in all animals were consistent with a minimal to slight local irritation due to the technical procedures.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Not sensitising

Executive summary:

METHOD:

The guinea pig maximisation test (GPMT) was chosen as test method used to evaluate skin sensitisation potential. The test was performed according to the OECD Guideline 406 (1992).

RESULTS:

Signs of irritation were noted during induction after application of sodium lauryl sulphate in both groups.

After challenge, the macroscopic and histopathological examinations revealed pathological lesion of delayed hypersensitivity in 1 out of the 20 treated animals. No noticeable cutaneous abnormality was noted in the 10 guinea-pigs examined in the control group.

CONCLUSION:

According to the CLP Regulation (EC n. 1272/2008), a substance is classified as skin sensitiser when in the Guinea pig maximisation test the response of at least 30 % of the animals is considered as positive.

In the present experiment, contrary to what it is possible to read in the concluding statement of the report (hypersensitivity in 1 out of the 20 treated animals), two of the animals tested showed a marked skin reaction, as described in the pathologist's report of the biopsies performed at 48 hours on the treated groups. This different in the evaluation of the effects does not affect the interpretation of the outcomes. The new percentage of sensitization reaction obtained (10 %) does not justify a classification as sensitising substance, according to the CLP Regulation (EC n. 1272/2008).