Disodium [3-[4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonato(3-)][1-[[2-hydroxy-5-(phenylazo)phenyl]azo]-2-naphtholato(2-)]chromate(2-)

Endpoint summary

Description of key information

Not sensitizer

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo study are available.

Species:

guinea pig

Strain:

Himalayan

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, 4414 Füllinsdorf/Switzerland
- Age at beginning of acclimatization period: 5 -7 weeks
- Body weight at beginning of acclimatization period: control and test group 291 - 374 g; pretest 292 - 364 g.
- Acclimatation: one week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretest. Only animals without any visual signs of illness were used for the study.
- Accommodation: individually in Makrolon type-3 cages (size: 22 x 37 x 15 cm) with autoclaved standard softwood bedding
- Diet: pelleted standard Kliba 342, Batch no. 63/94 guinea pig breeding/ maintenance diet ("Kliba", Klingentalmühle AG, CH-4303 Kaiseraugst), ad libitum.
- Water: community tap water from Füllinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1 g/l) via the drinking water.

ENVIRONMENTAL CONDITIONS
Air-conditioned with 10-15 air changes per hour and continuously monitored environment with a temperature between 21 and 25 °C, a relative humidity between 52 and 70 %, 12 hours artificial fluorescent light (approx. 100 Lux) /12 hours dark, music during the light period.

Route:

intradermal and epicutaneous

Vehicle:

water

Remarks:

bi-distilled

Concentration / amount:

PRETEST:
- Intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 5, 3 and 1 % of the test article in bi-distilled water.
- Epidermal application: 4 patches* of filter paper ( 2 x 2 cm) were saturated with the test article at
A = 25 % (this concentration used was found to be the most qualified to assure an optimum technical application procedure),
B = 15 %,
C = 10 % and
D = 5 %
of the test article in bi-distilled water and applied to the clipped and shaved flanks.

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Remarks:

bi-distilled

Concentration / amount:

PRETEST:
- Intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 5, 3 and 1 % of the test article in bi-distilled water.
- Epidermal application: 4 patches* of filter paper ( 2 x 2 cm) were saturated with the test article at
A = 25 % (this concentration used was found to be the most qualified to assure an optimum technical application procedure),
B = 15%,
C = 10% and
D = 5%
of the test article in bi-distilled water and applied to the clipped and shaved flanks.

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

Positive control results:

80 to 100 % of sensitized animals are usually obtained.

Remarks on result:

other: 1 animal out 20 showed positive reactions

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.

Interpretation of results:

other: Not classified according to the CLP Regulation

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.

The percentage of sensitization reaction obtained (10 %) does not justify a classification as sensitising substance, according to the CLP Regulation (EC n. 1272/2008).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

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).

The percentage of sensitization reaction obtained (10 %) does not justify a classification as sensitising substance, according to the CLP Regulation (EC 1272/2008).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In the CLP Regulation (EC n. 1272/2008), in the section 3.4 Respiratory or skin sensitisation, a skin sensitizer is defined as a substance that will lead to an allergic response following skin contact.

A substance shall be classified as skin sensitisers (Category 1) where data are not sufficient for sub- categorisation (1A and 1B) in accordance with the following criteria:

(a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons; or

(b) if there are positive results from an appropriate animal test ( according to 3.4.2.2.4.1).

If, in order to evaluate skin sensitisation potential, a Guinea pig maximisation test is performed, a substance is classified as skin sensitiser when the response of at least 30 % of the animals is considered as positive.

The percentage of sensitization reaction obtained (10 %) does not justify a classification as sensitising substance, according to the CLP Regulation (EC n. 1272/2008).