A mixture of: trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-(4-nitro-2-sulfonatoanilino)phenylazo)phenolato)ferrate(1-); trisodium bis(2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-nitro-2-sulfonatophenylazo)phenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(3-sulfonatophenylazo)phenolato)ferrate(1-); disodium 3,3'-(2,4-dihydroxy-1,3(or 1,5 or 3,5)-phenylenediazo)dibenzenesulfonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 14, 1990 to March 23, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1981

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study (different from LLNA test) is available.

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of: trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-(4-nitro-2-sulfonatoanilino)phenylazo)phenolato)ferrate(1-); trisodium bis(2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-nitro-2-sulfonatophenylazo)phenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(3-sulfonatophenylazo)phenolato)ferrate(1-); disodium 3,3'-(2,4-dihydroxy-1,3(or 1,5 or 3,5)-phenylenediazo)dibenzenesulfonate

EC Number:

406-870-1

EC Name:

A mixture of: trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-(4-nitro-2-sulfonatoanilino)phenylazo)phenolato)ferrate(1-); trisodium bis(2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(4-nitro-2-sulfonatophenylazo)phenolato)ferrate(1-); trisodium (2,4(or 2,6 or 4,6)-bis(3,5-dinitro-2-oxidophenylazo)-5-hydroxyphenolato)(2(or 4 or 6)-(3,5-dinitro-2-oxidophenylazo)-5-hydroxy-4(or 2 or 6)-(3-sulfonatophenylazo)phenolato)ferrate(1-); disodium 3,3'-(2,4-dihydroxy-1,3(or 1,5 or 3,5)-phenylenediazo)dibenzenesulfonate

Cas Number:

115100-55-5

Molecular formula:

not applicable

IUPAC Name:

triiron(3+) nonasodium 2-(2-{4-hydroxy-2-oxido-5-[2-(3-sulfophenyl)diazen-1-yl]phenyl}diazen-1-yl)-4,6-dinitrobenzen-1-olate 2-(2-{5-[2-(3,5-dinitro-2-oxidophenyl)diazen-1-yl]-2-hydroxy-4-oxidophenyl}diazen-1-yl)-4,6-dinitrobenzen-1-olate tris(2-(2-{5-[2-(3,5-dinitro-2-oxidophenyl)diazen-1-yl]-4-hydroxy-2-oxidophenyl}diazen-1-yl)-4,6-dinitrobenzen-1-olate) 2-{2-[4-hydroxy-5-(2-{4-[(4-nitro-2-sulfophenyl)amino]phenyl}diazen-1-yl)-2-oxidophenyl]diazen-1-yl}-4,6-dinitrobenzen-1-olate

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

Himalayan

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland
- Age at study initiation: ~ 11 weeks
- Weight at study initiation: 331 - 451 gram
- Housing: Group housing of 2 animals per cage with wire-mesh floors (ITL, Bergen, The Netherlands).
- Diet (e.g. ad libitum): Free access to standard guinea pig diet, including ascorbic acid (1600 mg/kg); LC 23-8, pellet diameter 4mm. (Hope Farms, Woerden, The Netherlands). In addition, once a week hay was provided (Broekman Institute, Someren, The Netherlands).
- Water (e.g. ad libitum): Free access to tap-water, diluted with decalcified water.
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 5 °C
- Humidity (%): 40-70 %
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was 12 hours artificial fluorescent light and 12 hours dark per day.

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

water

Concentration / amount:

Intradermal:
A) The test substance dissolved to 5% (w/w) with physiological saline.
B) Freunds' Complete Adjuvant (FCA, Oifco, Detroit, U.S.A.)f 50:50 with distilled water for injection. (pyrogen free, Ferensius AG, Bad Homburg, FRG).
C) The test substance, at twice the concentration usad in (A), emulsified in a 50:50 mixture of Freunds' Complete Adjuvant.
Epidermal:
0.5 ml of the test substance (50 % (w/w) in milli-RO water)

Day(s)/duration:

Intradermal injections: On day 1; Epicutaneous: Seven days after the intradermal injections,

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

0.05 ml of substance:
a = 25 % in milli-RO water.
b = 10 % in milli-RO water.
c = 5 % in milli-RO water.
d = milli-RO water.

Day(s)/duration:

two weeks after the epidermal induction application/24 hours

No. of animals per dose:

RANGE FINDING TESTS:
Induction
1 per sex per dose
Challenge
4 per sex per doses
MAIN TEST:
Test groups:
20 per sex per dose
Control group:
10 per sex per dose

Details on study design:

RANGE FINDING TESTS:
-INDUCTION EXPOSURE: intradermal
- No. of exposures: Four intradermal injections (0.1 ml/site)
- Test groups: 1 females guinea pigs
- Site: clipped shoulder region
- Frequency of applications: four injections
- Duration: single injection
- Concentrations:
Test group: 5 % (w/w) of the test substance in milli-RO water.
- Evaluation (hr after challenge): 24 and 48 hours later
The following parameters were recorded:
Erythema, Necrosis, Diameter:

-INDUCTION EXPOSURE: epicutaneous
-CHALLENGE EXPOSURE
- No. of exposures: one
- Exposure period: 24 hours.
- Test groups: 1 females guinea pigs
- Site: shaved left flank
- Concentrations:
Test group: 0.5 ml of a 50 % (w/w) concentration of the test substance in milli-RO water
- Evaluation (hr after challenge): 24 and 48 hours after removal of the dressings

-CHALLENGE EXPOSURE
- No. of exposures: one
- Exposure period: 24 hours.
- Test groups: 4 females guinea pigs
- Site: shaved left flank
- Concentrations:
Test group: 50 %, 25 %, 10 % and 5 % (w/w) test substance concentrations in milli-RO water (0.05 ml / concentration)
- Evaluation (hr after challenge): 24 and 48 hours after removal of the dressings

MAIN STUDY
A1. INDUCTION EXPOSURE: epicutaneous
- No. of exposures: Three pairs of intradermal injections (0.1 ml/site)
- Exposure period: day 1
- Test groups: 20 females guinea pig
- Control group: 10 females guinea pig
- Site: shaved left flank 4 X 6 cm
- Frequency of applications:
- Duration:
- Concentrations:
Test group:
A)5% (w/w) with physiological saline
B)Freunds' Complete Adjuvant (FCA, Oifco, Detroit, U.S.A.)f 50:50 with distilled water for injection. (pyrogen free, Ferensius AG, Bad Homburg, FRG).
C)The test substance, at twice the concentration usad in (A), emulsified in a 50:50 mixture of Freunds' Complete Adjuvant.

A2. INDUCTION EXPOSURE: epidermal
- No. of exposures: one
- Day(s) of challenge: Seven days after the intradermal injections
- Exposure period: 48 hours
- Test groups: 20 females guinea pig
- Control group: 10 females guinea pig
- Site: the scapular area (approximately 6 X 8 cm) was again clipped and shaved free of hair.
- Concentrations: A 2 x 4 cm patch of Metalline mounted on Micropore tape was treated with 0.5 ml of the test substance (50 % (w/w) in milli-RO water), the control group only with vehicle.
- Evaluation (hr after challenge): Reaction sites were assessed for erythema and oedema immediately after removal of the dressings.

B. CHALLENGE EXPOSURE
- No. of exposures: 4
- Day(s) of challenge: two weeks after the epidermal induction application.
- Exposure period: after approximately 24 hours.
- Test groups: 20 females guinea pig
- Control group: 10 females guinea pig
- Site: 5 x 5 cm area on the left flank
- Concentrations: A volume of 0.05 ml of each concentration
a = 25 % in railli-RO water.
b = 10 % in milli-RO water.
c = 5 % in milli-RO water.
d = milli-RO water.

Positive control substance(s):

yes

Remarks:

FORMALDEHYDE

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 %

No. with + reactions:

17

Total no. in group:

20

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10 %

No. with + reactions:

7

Total no. in group:

20

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5 %

No. with + reactions:

3

Total no. in group:

20

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

10

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.25 %

No. with + reactions:

13

Total no. in group:

20

Results 6

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.1 %

No. with + reactions:

3

Total no. in group:

20

Any other information on results incl. tables

SIGNS OF TOXICITY

No signs of systemic toxicity were observed during the primary irritation experiments, with exception of body weight loss in three of the five animals.

The choice of milli-RO water as vehicle in this test was based on the following:

- the test article dissolved\suspended well in milli-RO water.

- the test article is stable in milli-RO water.

In accordance with Magnusson and Kligman (1969) and based on the findings in the primary irritation experiments, the following concentrations were selected for the induction and challenge phase:

Intracutaneous induction: 5 % (w/w) in milli-RO water.

Epicutaneous induction: 50 % (w/w) in milli-RO water.

Challenge:

a = 25 % (w/w) in milli-RO water.

b = 10 % (w/w) in milli-RO water.

c = 5 % (w/w) in milli-RO water.

d = milli-RO water.

INDUCTION

Three experimental animals showed skin irritation after the 48 hours occluded epicutaneous induction exposure

CHALLENGE

CONTROL GROUP:

No positive skin reactions were evident after the challenge exposure

EXPERIMENTAL GROUP:

Four animals showed a positive reaction in response to the 25% concentration.

Thirteen other animals showed red spots with or without scaliness in response to one, two or three of the concentrations tested

TOXICITY SYMPTOMS / MORTALITY

No symptoms of systemic toxicity were observed in the animals during the study.

No mortality occurred during the study.

BOOY WEIGHTS

The average body weight gain of experimental and control animals was similar.

Applicant's summary and conclusion

Interpretation of results:

other: classified as Skin Sens. 1B according to the CLP regulation (EC) no. 1272/2008

Conclusions:

The test article at concentration of 25 % in water is considered to be a skin sensitizer when described under the test conditions.

Executive summary:

A GPMT was performed in order to obtain information on the potential of the test substance to induce delayed contact hypersensitivity (skin sensitisation) in the guinea pig, according to OECD guideline 406 (1981).

The intracutaneous route of administration (first induction at 5 %) was selected in order to obtain optimal contact between the test substance and elements of the immunosystem. Freunds Complete Adjuvant (FCA) was added to attract circulating immunocompetent cells to the injected area. The dermal route of administration (second induction and challenge) was performed. The test and control guinea pigs were challenged two weeks after the epidermal induction application. The dressings and residual test substance were removed after approximately 24 hours. The sites were assessed for redness and sweiling 24 and 48 hours after removal of the dressings. A positive control was tested in a concurrent study in order to verify the test validity. The following results were obtained:

POSITIVE SKIN REACTIONS TO THE CHALLENGE

	Test Substance Concentration
	25%	10%	5%	0%
EXPERIMENTAL GROUP Number of animals with positive reaction	4	0	0	0
Sensitisation rate	20	0	0	0
CONTROL GROUP Number of animals with positive reaction	0	0	0	0