Reaction products of 4,4'-Azo-3-hydroxynaphthalene-1-sulphonate coupled with 2-naphthol and 2-amino-4 (or 5)-nitrophenol, chelated with chromium (3+), sodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12.06. - 14.07.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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:

The test substance is skin irritant, due to the test Skin Sensitisation in Guinea Pigs was performed.

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen sodium [3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

EC Number:

267-824-2

EC Name:

Hydrogen sodium [3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

Cas Number:

67939-06-4

Molecular formula:

C36H20CrN5O9SNa

IUPAC Name:

Hydrogen sodium [3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)][1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphtholato(2-)]chromate(2-)

impurity 1

Reference substance name:

Sodium chloride

EC Number:

231-598-3

EC Name:

Sodium chloride

Cas Number:

7647-14-5

Molecular formula:

ClNa

IUPAC Name:

sodium chloride

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: BFA

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS- Source: Luboš Sobota, 298 03 Městec Králové, monitored breeding farm, RČH CZ 21760039- Age at receiving: 6-7 weeks - Weight at study initiation: 354.93 ± 32.82- Housing: sterilized shavings of soft wood, monitored conditions, microbiologically defined background, according to internal SOP No.40- Diet: pelleted standard diet for guinea pigs ad libitum (Altromin 3023 for Giunea pig)- Water: drinking tap water ad libitum (supplemented by ascorbic acid)- Acclimation period: 5 days- Number/Sex: 33 animals Pilot experiment: 3 animalsExposed group: 20 animalsControl group: 10 animals (13 males + 17 females)ENVIRONMENTAL CONDITIONSMicroclimatic conditions: - Room temperature 22 ± 3°C, permanently monitored- Relative humidity 30 – 70 %, permanently monitored- Light: 12 hour light/dark cycle: 6am-6pm/6pm-6am

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

other: 2-Mercaptobenzothiazole

Concentration / amount:

0.1 ml

Day(s)/duration:

0 - 6

Challenge

Route:

epicutaneous, occlusive

Vehicle:

other: vaseline

Concentration / amount:

5% test substance in vaseline

Day(s)/duration:

20

No. of animals per dose:

33 animals:Pilot experiment: 3 animalsExposed group: 20 animalsControl group: 10 animals

Details on study design:

MAIN STUDYDose levelsThe appropriate suspensions of the test substance determined from pilot experiment were as follows:Induction-intradermal injections:5% suspension of the test substance in physiological salineInduction-topical application:50% test substance in vaselineChallenge-topical application:5% test substance in vaselineA. INDUCTION EXPOSUREInduction: Intradermal InjectionsDay 0 – treated groupThree pairs of intradermal injections of 0.1 mL volume were given in the shoulder region, which was cleared of hair so that one of each pair lies on each side of the midline.Injection 1: a 1:1 mixture (v/v) Freunds Complete Adjuvant (FCA)/ physiological salineInjection 2: 5% suspension of the test substance in physiological salineInjection 3: 5% suspension of the test substance in a 1:1 mixture (v/v) FCA/physiological salineDay 0 – control groupThree pairs of intradermal injections of 0.1 mL volume were given in the same sites as in the treated animals.Injection 1: a 1:1 mixture (v/v) Freunds Complete Adjuvant (FCA)/ physiological salineInjection 2: physiological salineInjection 3: physiological saline in a 1:1 mixture (v/v) FCA/ physiological salineInduction: Topical Application Day 6 - treated group24 hours before application (day 5). A filter paper (2 x 4 cm) with 50% test substance in vaseline was applied to the test area and held in contact by an occlusive dressing for 48 hours. Day 6 – control group24 hours before application (day 5) the test area was cleared of hair. A filter paper (2 x 4 cm) with vaseline only was applied in a similar manner to the test area and held in contact by an occlusive dressing for 48 hours.B. CHALLENGE EXPOSURE- No. of exposures: 1- Day(s) of challenge: Day 20- Exposure period: 24h- Test groups: test substance in vaseline, vaseline- Control group: test substance in vaseline, vaseline- Site: L flank/R flank- Concentrations: 5% substance in vaseline- Evaluation (hr after challenge): 48h and 72hOBSERVATIONS:Clinical signs of intoxication and health condition: dailyMortality/viability: dailyBody weight: 0 and 24th dayThe skin reaction: 24, 48, 72, 96 and 120 hours after intradermal injection48, 72, 96, 120 hours after induction – topical application48 and 72 hours after challenge

Positive control substance(s):

yes

Remarks:

2-Mercaptobenzothiazole

Results and discussion

Positive control results:

Reliability of the experimental technique is checked periodically in about six-month interval by the experiment with known sensitiser 2-Mercaptobenzothiazole. The result of last experiment (March 2017): positive skin reaction – 7 animals (total number of animals in exposed group = 10), i.e. 70 %. According to the guideline the minimal number of animals with positive skin reaction should be 30 %. The result is satisfactory.The results of reference study are described in Study No. 413/15/6MK- Ref/03/2017: 2-Mercaptobenzothiazole - Senzibilizace kůže, VUOS-CETA Report No. 17-184.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5% test substance in vaseline

No. with + reactions:

6

Total no. in group:

20

Clinical observations:

In the course of experiment the animals did not show clinical symptoms of intoxication.

Remarks on result:

positive indication of skin sensitisation

Remarks:

discrete or patchy erythema

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

5 % test substance in vaseline

No. with + reactions:

4

Total no. in group:

20

Clinical observations:

In the course of experiment the animals did not show clinical symptoms of intoxication.

Remarks on result:

positive indication of skin sensitisation

Remarks:

discrete or patchy erythema

Results 3

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

vaseline only

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

In the course of experiment the animals did not show clinical symptoms of intoxication.

Remarks on result:

no indication of skin sensitisation

Results 4

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

vaseline only

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

In the course of experiment the animals did not show clinical symptoms of intoxication.

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

Table No. 3: Grading of skin reactions

Exposed				Control
Animal No.	Sex	Start of study	End of study	Animal No.	Sex	Start of study	End of study
11	♂	384.09	520.20	1	♂	422.18	692.90
12	♂	343.95	498.15	2	♂	305.90	470.39
13	♀	331.30	504.91	3	♂	367.05	615.72
14	♀	329.58	519.66	4	♂	434.94	632.96
15	♀	321.99	468.25	5	♂	331.98	584.92
16	♀	349.46	518.55	6	♂	338.86	488.15
17	♀	354.11	488.76	7	♂	375.87	549.48
18	♀	313.27	505.37	8	♀	410.87	544.77
19	♀	352.17	502.38	9	♀	443.82	616.87
20	♀	379.33	536.28	10	♀	375.20	525.78
21	♀	389.73	523.80
22	♀	409.95	536.91
23	♀	324.51	512.13
24	♀	383.07	552.18
25	♀	318.42	507.17
26	♀	327.13	493.86
27	♂	426.25	579.36
28	♂	383.19	577.26
29	♂	325.11	535.23
30	♂	352.03	563.61

♂ – male

♀ – female

0/0 –erythema/oedema

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test substance, Acid Black 227, caused a positive skin reaction in 7 of 20 animals, i.e. in 35 % of animals, which were exposed to the test substance. Therefore the sensitisation response is positive acc. to criteria in chap. 3.10 of final report.The body weight of animals increased through the study and it was not affected by the treatment. The exposed animals showed no other negative clinical symptoms throughout the experiment.The test substance, Acid Black 227, was shown to be a contact allergen in guinea pigs.

Executive summary:

The test substance, Acid Black 227, was tested for the assessment of skin allergic effects using albino guinea pigs (strain BFA).

The test was performed according to the EU Method B.6, Skin sensitisation. The method is analogous to the OECD Test Guideline No. 406, Skin Sensitisation.

The Magnusson and Kligman maximization procedure was followed. The pilot experiment was implemented on 3 animals. The main test was performed on 20 treated and 10 control animals.

The experiment proceeded in three phases: two induction phases (intradermal injections and topical application) and the challenge phase. Potential skin reactions were evaluated at the end of experiment.

The evaluation of skin reactions in the exposed group carried out at 48 hours after the start of the challenge phase showed discrete erythema in 6 animal in left flank applied with the test substance. Evaluation at 72 hours after the start of the challenge phase of study showed discrete erythema in next 1 animal in left flanks applied with the test substance and in 3 animals the discrete erythema persisted from the previous day.

The body weight of animals increased through the study and it was not affected by the treatment. The exposed animals showed no other negative clinical symptoms throughout the experiment.

The test substance, Acid Black 227, caused a positive skin reaction in 7 of 20 animals, i.e. in 35 % of animals, which were exposed to the test substance. Therefore the sensitisation response is positive. The test substance, Acid Black 227, was shown to be a contact allergen in guinea pigs.